Modulation of integrin-mediated signal transduction

ABSTRACT

The present invention discloses that phosphorylation of cytoplasmic tyrosine residues in the β-subunit of integrins is needed for signal protein association. The invention provides methods of identifying signaling partners involved in integrin mediated signaling, methods of identifying agents which block integrin mediated signaling, methods of using agents which block integrin mediated signaling to modulate biological and pathological processes, and agents which block integrin mediated signaling.

RELATED APPLICATION

[0001] This application claims priority from U.S. provisionalapplication Serial No. 60/005,567, filed Oct. 18, 1995, the contents ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the modulation ofintegrin-mediated signal transduction, particularly signal transductionmediated by GPIIb-IIIa on platelets. The invention relates specificallyto the identification of molecules that mediate integrin signaling andto methods of modulating integrin-mediated signaling.

BACKGROUND OF THE INVENTION

[0003] Integrins are a family of αβ heterodimers that mediate adhesionof cells to extracellular matrix proteins and to other cells (Clark etal., Science (1995) 268: 233-239). Integrins also participate in signaltransduction, as evidenced by either an alteration in adhesive affinityof cell surface integrins in response to cellular activation (termedinside-out signal transduction) or by affecting intracellular signalingpathways following integrin-mediated adhesion (termed outside-in signaltransduction). Many biological responses are dependent at least to someextent upon integrin-mediated adhesion and cell migration, includingembryonic development, hemostasis, clot retraction, mitosis,angiogenesis, cell migration, inflammation, immune response, leukocytehoming and activation, phagocytosis, bone resorption, tumor growth andmetastasis, atherosclerosis, restenosis, wound healing, viralinfectivity, amyloid toxicity, programmed cell death and the response ofcells to mechanical stress.

[0004] The integrin family consists of 15 related known a subunits (α1,α2, α3, α4, α5, α6, α7, α8, α9, αE, αV, aIIb, aL, aM, and aX) and 8related known β subunits (β1, β2, β3, β4, β5, β6, β7, and β8, Luscinskaset al., FASEB J (1994) 8:929-938). Integrin α and β subunits are knownto exist in a variety of pairings as indicated in FIG. 1. Integrinligand specificity is determined by the specific pairing of the α and βsubunits, although some redundancy exists as several of the integrinsare known to bind the same ligand. FIG. 2 shows the sequences of thecytoplasmic domains of GPIIb and GPIIIa, including the cytoplasmicdomains of other α and β subunits, respectively, that have homologouscytoplasmic domains. Most integrins containing the β1, β2, β3, β5, β6,and β7 subunits have been found to transduce signals (reviewed by Hynes,Cell (1992) 62:11-25). Integrins are involved in both “inside-out” and“outside-in” signaling events.

[0005] Various pathologies associated with integrin-related defects areknown. For example, inherited deficiencies of GPIIb-IIIa (also termedαIIβ3) content or function have been described (termed Glanzmann'sthrombasthenia) and are characterized by platelets that do not bindadhesive proteins and therefore fail to aggregate, resulting in alife-long bleeding diathesis. Inhibitors of the binding of fibrinogenand von Willebrand factor to GPIIb-IIIa have been described and havebeen found to block platelet aggregation in vitro and to inhibitclinical thrombosis in vivo (The EPIC Investigators, New England Journalof Med (1994) 330:956-961; Tcheng, J. E. et al., Circulation (1995)91:2151-2157). Also, leukocyte 2 0 adhesion deficiency (LAD) resultsfrom the absence of a β2 subunit.

[0006] A. Inside-Out Signaling

[0007] Inside-out signal transduction has been observed for β1, β2, andβ3 integrins. (Hynes, R. O. Cell (1992) 69:11-25; Phillips, D. R. etal., Cell (1991) 65:359-362; Smyth, S. S. et al., Blood (1993)81:2827-2843; Ginsberg, M. H. et al., Thromb Haemostasis (1993)70:87-93; Juliano, R. L. et al., Cell Biol (1993) 120:577-585;Rouslahti, E., J Clin Invest (1991) 87:1-5.

[0008] Perhaps the most widely studied integrin that is involved ininside-out signaling is GPIIb-IIIa, the receptor for the four adhesiveproteins, fibrinogen, von Willebrand factor, vitronectin andfibronectin, on stimulated platelets (Phillips, D. R. et al., Blood(1988) 71:83143). The binding of adhesive proteins to GPIIb-IIIa isrequired for platelet aggregation and normal hemostasis and is alsoresponsible for occlusive thrombosis in high shear arteries.

[0009] GPIIb-IIIa is known to be involved in inside-out signaltransduction because GPIIb-IIIa on the surface of unstimulated plateletsis capable of recognizing only immobilized fibrinogen. In response toplatelet stimulation by agents such as thrombin, collagen and ADP,GPIIb-IIIa becomes a receptor for the four adhesive proteins in theprevious paragraph, and the binding of fibrinogen and von Willebrandfactor causes platelets to aggregate. A monoclonal antibody has beendescribed which detects the activated, receptor competent state ofGPIIb-IIIa, suggesting that the conformation of the receptor competentform of GPIIb-IIIa differs from that of GPIIb-IIIa which does not bindsoluble fibrinogen or von Willebrand factor (Shattil, S. J. et al., JBiol Chem (1985) 260:11107-11114). It has been postulated thatinside-out GPIIb-IIIa signal transduction is dependent on cellularproteins that act to repress or stimulate GPIIb-IIIa activation(Ginsberg, M. H. et al., Curr Opin Cell Biol (1992) 4:766-771).

[0010] β2 integrins on leukocytes also respond to inside-out signaltransduction which accounts, for example, for the increased bindingactivity of LFA-1 on stimulated lymphocytes and the increased bindingactivity of MAC-1 on stimulated neutrophils (reviewed by Springer, T.,Curr Biol (1994) 4:506-517).

[0011] B. Outside-In Signaling

[0012] Most integrins can be involved in outside-in signal transductionas evidenced by observations showing that binding of adhesive proteinsor antibodies to integrins affects the activities of many cells, forexample cellular differentiation, various markers of cell activation,gene expression, and cell proliferation (Hynes, R. O. Cell (1992)69:11-25). The involvement of GPIIb-IIIa in outside-in signaling isapparent because the binding of unstimulated platelets to immobilizedfibrinogen, a process mediated by GPIIb-IIIa, leads to plateletactivation and platelet spreading (Kieffer, N. et al., J Cell Biol(1991) 113:451-461).

[0013] Outside-in signaling through GPIIb-IIIa also occurs duringplatelet aggregation. Signaling occurs because fibrinogen or vonWillebrand factor bound to the activated form of GPIIb-IIIa on thesurface of stimulated platelets, coupled with the formation ofplatelet-platelet contacts, causes further platelet stimulation throughGPIIb-IIIa signal transduction. In this manner, binding of adhesiveproteins to GPIIb-IIIa can both initiate platelet stimulation or canaugment stimulation induced by the other platelet agonists such as ADP,thrombin and collagen. The binding of soluble fibrinogen to GPIIb-IIIaon unstimulated platelets can also be induced by selected GPIIb-IIIaantibodies such as LIBS6 (Huang, M -M. et al., J Cell Biol (1993)122:473-483): although platelets with fibrinogen bound in this mannerare not believed to be stimulated, such platelets will aggregate ifagitated and will become stimulated following aggregation throughGPIIb-IIIa signal transduction.

[0014] Outside-in integrin signal transduction results in the activationof one or more cascades within cells. For GPIIb-IIIa, effects caused byintegrin ligation include enhanced actin polymerization, increasedNa⁺/H⁻ exchange, activation of phospholipases, increased phosphatidylturnover, increased cytoplasmic Ca⁺⁺, and activation of kinases. Kinasesknown to be activated include PKC, myosin light chain kinase, src, sykand pp125FAK. Kinase substrates identified include pleckstrin, myosinlight chain, src, syk, pp125FAK, and numerous proteins yet to beidentified (reviewed in Clark, E. A. et al., Science (1995)268:233-239). Many of these signaling events, includingphosphorylations, also occur in response to ligation of other integrins(reviewed in Hynes, R. O. Cell (1992) 69:11-25). Although these otherintegrins have distinct sequences and distinct α-β parings that allowfor ligand specificity, the highly conserved nature of the relativelysmall cytoplasmic domains, both between species and between subunits,predicts that related mechanisms will be responsible for thetransduction mechanisms of many integrins.

[0015] C. Signal Transduction

[0016] Despite the numerous observations on the binding of cytoplasmicproteins to GPIIb-IIIa and other integrins, it is not yet known whetherthe binding of any of these is involved in integrin signal transductionnor is it known what regulates their binding to the integrin. There havebeen several attempts to determine whether phosphorylation of thecytoplasmic domain of GPIIIa and β1 is responsible for GPIIb-IIIa signaltransduction. These studies, discussed below, apparently originated fromthe suggestion made following the determination of the primary sequenceof GPIIIa and PI which showed that tyrosine 747 on the cytoplasmicdomain of GPIIIa (and tyrosine 788 on the cytoplasmic domain of β1) wasa possible phosphorylation site as it existed within a motif similar toa tyrosine which exists in the cytoplasmic domains of the epidermalgrowth factor and insulin receptors which are known phosphorylationsites (Fitzgerald, L. et al., J Biol Chem (1987) 262:3936-3939; Tamkun,J. W. et al., Cell (1986) 46:271-282).

[0017] Nonetheless, the involvement of the cytoplasmic domain ofGPIIb-IIIa in integrin signal transduction is inferred from mutagenesisexperiments. Deletion of the cytoplasmic domain of GPIIb results in aconstitutively active receptor that binds fibrinogen with an affinityequivalent to the wild-type complex, implying that the cytoplasmic tailof GPIIb has a regulatory role (O'Toole, T. E. et al., Cell Regul (1990)1:883-893). Point mutations, deletions and other truncations ofGPIIb-IIIa affects the ligand binding activity of GPIIb-IIIa and itssignaling response (Hughes, P. E. et al., J Biol Chem (1995) 270:12411-12417; Ylanne, J. et al., J. Biol Chem (1995) 270:9550-9557).

[0018] Chimeric, transmembrane proteins containing the cytoplasmicdomain of GPIIIa, but not of GPIIb, inhibit the function of GPIIb-IIIa(Chen, Y. -P. et al., J Cell Biol (1994) 269:18307-18310), implying thatfree GPIIIa cytoplasmic domains bind proteins within cells and that thisbinding is necessary for normal GPIIb-IIIa function. Several proteinshave been shown to bind either the transmembrane domains or thecytoplasmic domains of GPIIb or GPIIIa.

[0019] CD 9, a member of the tetraspanin family of proteins (Lanza, F.et al., J Biol Chem (1991) 266:10638-10645), has been found to interactwith GPIIb-IIIa on aggregated platelets. 3-endonexin, a proteinidentified through two hybrid screening using the cytoplasmic domain ofGPIIIa as the “bait”, has been found to interact directly andselectively with the cytoplasmic tail of GPIIIa (Shattil, S. et al.,Throm and Haemost (1995) 73:1190). β3-endonexin shows decreased bindingto the GPIIIa cytoplasmic domain containing the thrombasthenic S752-Pmutation. It is not yet known whether either of these GPIIIa-bindingproteins are involved in signal transduction.

[0020] Cytoplasmic proteins that bind to αVβ3 have also been describedwhich may be interacting with the integrin at the GPIIIa cytoplasmicdomain sequence. Bartfeld and coworkers (Bartfeld, N. S. et al., J BiolChem (1993) 268:17270-17276) used immunoprecipitation from detergentlysates to show that a MW=190 kDa protein associates with the αVβ3integrin from PDGF-stimulated 3T3 cells. IRS-1 was found to bind to theαV 3 integrin following insulin stimulation of Rat-1 cells stablytransfected with DNA encoding the human insulin receptor (Vuori, K. etal., Science (1994) 266:1576-1578). β1-containing hybrid proteins alsohave a dominant negative effect on integrin function implying that β1integrins also bind cytoplasmic proteins (LaFlamme, S. E. et al., J CellBiol (1994) 126:1287-1298). The importance of the cytoplasmic domain ofβ1 is underscored by the demonstration that its removal markedly reducesthe adhesive activity of the integrin α5⊕1 (Hyashi, Y. et al., J CellBiol (1990) 110:175-184; Ylanne, J., et al., J Cell Biol (1993)122:223-233). Mutations of defined sequences of the cytoplasmic domainof β1 have been shown to decrease integrin recruitment to adhesionplaques (Reszka, A. A. J Cell Biol (1992) 117:1321-1330). Several β1cytoplasmic domain binding proteins have been described. Otey andcoworkers (Otey, C. A. et a!, J Biol Chem (1993) 268:21193-21197) haveused synthetic peptides to map the binding site for α-actinin within thecytoplasmic domain of β1. Talin binding to a peptide corresponding tothe cytoplasmic domain of β1 has been observed (Horwitz, A., et al.,Nature (1986) 320:531-533). Argraves and coworkers (Argraves, W. S. etal., Cell (1989) 58:623-629) also used synthetic peptides to show thatfibulin bound to the cytoplasmic domain of β1. The NH₂-terminal,noncatalytic domain of pp125FAK has been found to directly bind to thecytoplasmic tail of β1 and to recognize integrin sequences distinct fromthose involved in binding to α-actinin (Schaller, M. D. et al., J CellBiol (1995) 130:1181-1 187). Integrin-associated kinase (IAK) is atyrosine kinase that has been found to bind to the cytoplasmic tail ofβ1 (Hannigan, G. E. et al., Nature (1996) 379:91-96).

[0021] In order to determine whether or not GPIIIa was phosphorylated ontyrosine residues as a consequence of platelet activation, the followingexperiments were performed. GPIIb-IIIa from control andthrombin-stimulated platelets was analyzed for changes inphosphorylation and it was observed that stimulation caused an increasein the phosphorylation of GPIIIa, but that the phosphorylation wasprimarily on serine, with no detectable phosphorylation of tyrosine(Parise, L. V. et a., Blood (1990) 75:2363-2368). Consistent with thesefindings, a variant of Glanzmann's thrombasthenia has been describedwhere the deficiency of the platelet aggregation response has beenattributed to the replacement of a serine residue in the cytoplasmictail of GPIIIa by a proline residue (Chen, Y. -P. et al., Proc Natl AcadSci USA (1992) 89:10169-10173). This implies that the sequence thatoccurs for normal GPIIIa is required for GPIIb-IIIa signal transduction,possibly involving the activation of the receptor function.

[0022] Other studies have shown that the sequences of the cytoplasmicdomains of GPIIIa, β1 and β2 which contain tyrosines are important fornormal functioning of GPIIb-IIIa and of other integrins. Substitution oftyrosine 747 by alanine in GPIIIa transfected into CHO cells abolishedGPIIIa-mediated cell spreading, blocked the recruitment of GPIIb-IIIa topreestablished adhesion plaques, and decreased the ability of GPIIb-IIIato mediate internalization of fibrinogen-coated particles (Ylanne, J. etal., J Biol Chem (1995) 270:9550-9557). Additional experiments in thisstudy showed further that substitution of tyrosine 759 by alaninedecreased cell spreading and the recruitment of GPIIb-IIIa to plaques,while deletion of the carboxy terminal pentapeptide that contains thissequence had an even more pronounced effect on the function of theintegrin. These authors concluded integrin-mediated cell spreading doesnot occur because the factors that are absolutely required forintegrin-mediated cell spreading cannot bind either the GPIIIa truncatedat residue 757 or to the integrin with tyrosine 747 on GPIIIasubstituted by alanine. Point mutations in homologous domains in β1- andβ2-containing integrins also suggest that these domains are functionalas these mutations affect integrin-cytoskeletal interactions (Reszka, A.A. et al., J Cell Biol (1992) 117:1321-1330) and integrin activation(Hibbs, M. L. et al., J Exp Med (1991) 174:1227-1238), respectively.Similarly, an NPXY motif in the integrin β3 cytoplasmic subunit tailappears necessary for melanoma cell migration (Filardo, E. J. et al., JCell Biol (1995) 130:441-450; O'Toole, T. E. J Biol Chem (1995)270:8553-8558).

[0023] Some commentators have suggested that the phosphorylation ofisolated cytoplasmic tyrosine residues was implicated in signaltransduction. For example, GPIIb and IIIa isolated from human plateletswere reported to serve as substrates for PP60^(c-src) (Findik, D. etal., FEBS (1990) 262:1-4). The tyrosine(s) on the cytoplasmic domain ofGPIIIa also have been found to be an in vitro substrate for src (Elmore,M. A. et al., FEBS (1990) 269:283-287), but it has not been demonstratedthat src phosphorylates GPIIIa in vivo. Others have reported that thesetyrosine residues are not phosphorylated during normal integrin function(Hillery, C. A. et al., J Biol Chem (1991) 266:14663-14669). Thus,neither of the GPIIIa containing integrins, GPIIb-IIIa or αVβ3, arebelieved to be phosphorylated on tyrosine.

[0024] Tyrosine phosphorylation of β1 has been observed, however, butonly in cells overexpressing vSrc (Hirst, R. et al., PNAS USA (1986)83:6470-6474). β1 phosphorylation coincides with a decrease in theability of the α5β1 integrin to mediate cell adhesion (Horwitz, A. etal., Nature (1986) 320:531-533) and a decrease in the ability of thisintegrin to localize to focal adhesion plaques (Johansson, M. W. et al.,J Cell Biol (1994)126:1299-1309). Increased phosphorylation of thecytoplasmic domain of β1 may decrease the binding of talin (Tapley, P.et al., Oncogene (1989) 4:325-333). The available data on β1 integrinssuggest that tyrosine phosphorylation of β1 has a negative effect on itsfunction and that 15-tyrosine phosphorylation of β1 may be associatedwith a transformation phenotype. Similarly, phosphorylation of twoconserved tyrosines in the cytoplasmic domain of the integrin βp subunitwas found to be unnecessary for developmental functions in Drosophila(Grinblat, Y. et al., Development (1994) 120:91-102).

[0025] Integrin binding to adhesive proteins and integrin signaltransduction have a wide variety of physiological roles, as identifiedabove. Enhanced signaling through integrins allows for increased celladhesion and activation of intracellular signaling molecules whichcauses enhanced cell mobility and growth, enhanced cell responsiveness,and modulations in morphological transformations. Although integrinsresponsible for cellular function have been described and signalingevents are beginning to be elucidated, the mechanism by which integrinstransduce signals remains to be determined. Identification of theevent(s) which allow for integrin interactions with cytoplasmicsignaling molecules will greatly enhance the understanding of integrinfunction and will provide for agents which can modulate integrinfunction. Such agents will be useful for the treatment and diagnosis ofa wide spectrum of pathologies, including the processes described above.The present invention describes the event which allows for theinteraction in vivo of GPIIb-IIIa with intracellular signaling moleculesand peptide structures which can be used to modulate these signalingevents.

SUMMARY OF THE INVENTION

[0026] The present invention is based in part on the discovery thattyrosine residue in the cytoplasmic domain of β-subunits of integrinsrequire phosphorylation in vivo for signal transduction. Signalingpartners become associated with phosphorylated cytoplasmic domains whileno association occurs with integrin subunits lacking phosphorylatedcytoplasmic tyrosine residues.

[0027] Based on these discoveries, the present invention providesphosphorylated peptide fragments of the cytoplasmic domains of integrinβ-subunits. These peptides can be used to isolate integrin signalingproteins and complexes, as agents to reduce the association of signalingpartners with integrins, and as targets for the development ofpharmaceutical agents.

[0028] The present invention further provides methods for reducing orblocking the association of an integrin with a cytoplasmic signalingpartner. Specifically, the association of an integrin with a cytoplasmicsignaling partner can be blocked or reduced by contacting an integrinhaving a phosphorylated tyrosine in the cytoplasmic domain of theβ-subunit, or a fragment thereof comprising the phosphorylatedcytoplasmic domain, with an agent which blocks the binding of thesignaling partner to the integrin. The method can use an agent whichbinds to the cytoplasmic domain of the integrin or an agent which bindsto the signaling partner.

[0029] Blocking integrin signaling partner associations can be used tomodulate biological and pathological processes which require an integrinmediated signal. Such methods and agents can be used to modulatecellular attachment or adhesion to a substrate or another cell, cellularmigration, cellular proliferation and cellular differentiation.Pathological processes involving these action include thrombosis,inflammation, tumor metastasis, wound healing and others noted above.

[0030] The present invention further provides methods for isolatingintegrin signaling partners. Integrin signaling partners are isolatedusing a tyrosine phosphorylated cytoplasmic domain of a β-subunit as acapture probe. Specifically, a peptide containing the phosphorylatedcytoplasmic tyrosine of an integrin is mixed with an extract preparedfrom an integrin expressing cell under condition which allow associationof the β-subunit fragment with a signaling partner. Non-associatedcellular constituents are removed from the mixture and the signalingpartner is released from the β-subunit probe. Signaling partnersisolated by this method are useful in preparing antibodies and alsoserve as targets for drug development.

[0031] The present invention further provides methods to identify agentswhich can block or modulate the association of an integrin with asignaling partner. Specifically, an agent can he tested for the abilityto block or reduce or otherwise modulate the association of an integrinwith a signaling partner by incubating a peptide comprising thephosphorylated cytoplasmic domain of the β-subunit of an integrin with asignaling partner and a test agent, and determining whether the testagent blocks or reduces the binding of the signaling partner to theintegrin peptide. Agonists, antagonists and other modulators expresslyare contemplated.

[0032] The present invention further provides methods of reducing theseverity of pathological processes which require integrin mediatedsignaling. Since phosphorylation is required for the association ofintegrins with cytoplasmic signaling partners agents which blockintegrin/signaling partner association, agents which block tyrosinephosphorylation, and agents which dephosphorylate phosphorylatedtyrosines can be used in therapeutic methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows pairing of a and p integrin subunits.

[0034]FIG. 2 shows cytoplasmic domains of various integrin subunits.

[0035]FIG. 3 shows tyrosine phosphorylation of the integrin GPIIIa (03)subunit.

[0036]FIG. 4 shows in vitro phosphorylation of GPIIIa (p3).

[0037]FIG. 5 shows the interaction of signaling protein SHC and GR B2with tyrosine phosphorylated β3 peptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0038] I. General Description

[0039] The present invention is based in part on identifying themechanism by which integrins are modified so that they can interact withsignaling proteins and signaling complexes. In the following Examples,data are provided showing that GPIIIa unexpectedly is phosphorylated ata tyrosine residue in the cytoplasmic domain during platelet aggregationand that src and fyn may be the responsible kinases. The high degree ofsequence and motif structure homology found around the phosphorylationsite of the cytoplasmic domain of GPIIIa when compared to thecorresponding structure/sequences found in other β subunits,particularly β1, β5, β6 and β7 subunits, predicts that tyrosinylphosphorylation is a widely utilized mechanism for regulating signalprotein association within the integrin family of receptors.

[0040] The present invention is further based on identifying how tomodify fragments of the β subunit of an integrin so that they interactwith integrin associated signaling proteins. In the Examples,phosphorylated peptides corresponding to part of the cytoplasmic domainof an integrin, for example, GPIIIa, are demonstrated to, bindcytoplasmic signaling proteins and complexes while the unphosphorylatedpeptide did not. The phosphorylated peptides also bound to additionalproteins of unknown identity. The phosphorylated peptides can be used asan agent, or serve as a target for agents, which can be used to inhibitintegrin mediated signaling, for example to inhibit biological processesrequiring GPIIb-IIIa or αVβ3 signal transduction. Further,phosphorylated peptides corresponding to the homologous sequences in theβ1, β5, β6 and β7 subunits, or mimics of these sequences, can be used asan agent or serve as a target for agents which can be used to inhibitsignaling for other integrins containing these P subunits.

[0041] The present invention is further based on the development ofmethods for isolating integrin signaling proteins. Phosphorylatedpeptide probes based on the cytoplasmic domain of integrin β subunitsare used as capture probes to isolate integrin associated signalingproteins. Dominant negative proteins, DNAs encoding these proteins,antibodies to these signaling proteins, peptide fragments of theseproteins or mimics of these proteins may be introduced into cells toaffect integrin function. Additionally, these proteins provide a noveltarget for screening of synthetic small molecules and combinatorial ornaturally occurring compound libraries to discover novel therapeutics toregulate integrin function.

[0042] Utilizing these observations, the present invention providesmethods of identifying tyrosine phosphorylation sites on integrin βsubunits, methods of identifying cytoplasmic signaling partners whichassociate with phosphorylated β subunits, methods to assay for integrinmediated signaling, methods to identify agents which block integrinmediated cytoplasmic signaling, and therapeutic uses for agents whichmodulate integrin mediated cytoplasmic signaling.

[0043] II. Specific Embodiments

[0044] A. Tyrosine Phosphorylation Sites in the Cytoplasmic Domain ofIntegrin βSubunits

[0045] In the Examples, data are presented which demonstrate that thetyrosine residues found within the cytoplasmic domain of the β subunitof an integrin are phosphorylated in vivo. Upon phosphorylation,cellular signaling partners found within the cytoplasm of theintegrin-expressing cell become associated with the β subunit. Thesesignaling partners do not become associated with cytoplasmic domains ofβ subunits which lack a phosphorylated tyrosine. Based on theseobservations, one aspect of the present invention provides, as examples,the amino acid sequence of the cytoplasmic domain of integrin β subunitswhich, upon phosphorylation, become associated with signaling proteinsand complexes.

[0046] In detail, the present invention provides six β subunit peptidespossessing a tyrosine residue which can be phosphorylated by a cellulartyrosine kinase. The cytoplasmic domain sequences of each of thecharacterized subunits, with the corresponding phosphorylation siteidentified, are: β1 subunit:NH-D-T-G-E-N-P-I-Y(PO₃)-K-S-A-V-T-T-V-V-N-P-K-Y(PO₃)-E-G-K-COOH β2subunit:NH-D-L-R-E-Y(PO₃)-R-R-F-E-K-E-K-L-S-Q-W-N-N-D-N-P-L-F-K-S-A-T-COOH β3subunit: NH-D-T-A-N-N-P-L-Y(PO₃)-K-E-A-T-S-T-F-T-N-I-T-Y(PO₃)-R-G-T-COOHβ5 subunit:NH-E-M-A-S-N-P-L-Y(PO₃)-R-K-P-I-S-T-H-T-V-D-F-T-F-N-K-F-N-K-S-Y(PO₃)-N-G-T-V-D-COOHβ6 subunit:NH-Q-T-G-T-N-P-L-Y(PO₃)-R-G-S-T-S-T-F-K-N-V-T-Y(PO₃)K-H-R-E-K-Q-K-V-D-L-S-T-D-C-COOHor NH-Q-T-G-T-N-P-L-Y(PO₃)-R-G-S-T-S-T-F-K-N-V-T-Y(PO₃)-K-H-R-COOH β7subunit:NH-D-R-R-E-Y(PO₃)-S-R-F-E-D-K-Q-Q-Q-L-N-W-K-Q-D-S-N-P-L-Y(PO₃)-K-S-A-I-COOH

[0047] The present invention provides these peptides, as well as thoseof the foregoing section of this specification and the peptidesdisclosed in the examples that follow, as well as homologous portions ofallelic variants of the corresponding integrins, and conservative aminoacid substitutions of these peptides. As used herein, an allelic variantrefers a naturally occurring β integrin (or more specifically to itscytoplasmic domain) having a different amino acid sequence than thatspecifically recited above. Allelic variants, though possessing adifferent amino acid sequence than those recited above, will still havethe requisite phosphorylatable tyrosine residue recognized by theintegrin signaling partner and will function to associate or interactwith this partner as part of the relevant signaling cascade.

[0048] As used herein, a conservative amino acid substitution refers toalterations in the amino acid sequence which do not adversely effect thepeptide. A substitution is said to adversely effect the peptide when thealtered sequence prevents the phosphorylation of the peptide or theability of the phosphorylated peptide to associate with a signalingpartner. For example, the overall charge, structure orhydrophobic/hydrophilic properties of the peptide can be altered withoutadversely effecting the peptide. Accordingly, the amino acid sequence ofthe above peptides can be altered, for example to render the peptidemore hydrophobic or hydrophilic, without adversely effecting the abilityof the peptide to become phosphorylated or the ability of thephosphorylated peptide to associate with a signaling partner.

[0049] Ordinarily, the peptides and analogs thereof claimed herein willhave an amino acid sequence having at least 75% amino acid sequenceidentity with the disclosed peptides from the cytoplasmic domains of βintegrins (such as those disclosed in Examples 2 and 4-8), morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 95%. Identity or homology with respect to suchsequences is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the known peptides, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent homology, and not considering any conservativesubstitutions as part of the sequence identity. None of N-terminal,C-terminal or internal extensions, deletions, or insertions into thepeptide sequence shall be construed as affecting homology.

[0050] Thus, the claimed peptides and analog molecules that are thesubject of this invention include molecules having the sequencesdisclosed; fragments thereof having a consecutive sequence of at leastabout 3, 5, 10 or 15 amino acid residues from the correspondingcytoplasmic domains of β integrins; amino acid sequence variants of suchsequences wherein an amino acid residue has been inserted N- orC-terminal to, or within, the disclosed sequences or their fragments asdefined above; and amino acid sequence variants of the disclosedsequences or their fragments as defined above which have beensubstituted by another residue. Contemplated polypeptides include thosecontaining predetermined mutations by, e.g., homologous recombination,site-directed or PCR mutagenesis, and the corresponding cytoplasmicdomain polypeptides of other animal species, including but not limitedto rabbit, rat, murine, porcine, bovine, ovine, equine and non-humanprimate species, and the alleles or other naturally occurring variantsof the cytoplasmic domains of the integrins of the foregoing species andof human sequences; derivatives wherein the peptides or their fragmentshave been covalently modified, by chemical, enzymatic, or otherappropriate means, to attach a moiety other than a naturally occurringamino acid (for example a detectable moiety such as an enzyme orradioisotope); glycosylation variants (insertion of a glycosylation siteor deletion of any glycosylation site by deletion, insertion orsubstitution of appropriate amino acid); and soluble forms as well asirreversibly phosphorylated forms.

[0051] The novel proteins and peptides of the present invention arepreferably those which share a common biological activity with thedisclosed peptides, including but not limited to being phosphorylated bytyrosine kinases that phosphorylate native cytoplasmic domains of βintegrins, interactions with signaling partners of β integrincytoplasmic domains, other effector or receptor function orcross-reactive antigenicity. Such fragments and variants exclude any βintegrin cytoplasmic domain peptides heretofore made public, includingany known protein or polypeptide of any animal species, which isotherwise anticipatory under 35 U.S.C. §102 as well as polypeptidesobvious over such known protein or polypeptides under 35 U.S.C. §103.

[0052] As described below, these peptides can be used: 1) to identifyand isolate integrin signaling partners, 2) in methods to identifyagents which block the association of an integrin with a signalingpartner, 3) as a target to assay for integrin mediated signaling, and 4)as a therapeutic agent to block the association of an integrin with asignaling partner.

[0053] Nucleic acids encoding the foregoing peptides also arecontemplated as part of the present invention. For purposes of thisinvention, “nucleic acid” is defined as RNA or DNA that encodes apeptide as defined above, or is complementary to nucleic acid sequenceencoding such peptides, or hybridizes to such nucleic acid and remainsstably bound to it under stringent conditions, or encodes a polypeptidesharing at least 75% sequence identity, preferably at least 80%, andmore preferably at least 85%, with the peptide sequences. It istypically at least about 9 nucleotides in length, and specificallycontemplated are genomic DNA, cDNA, mRNA and antisense molecules, aswell as nucleic acids based on an alternative backbone or includingalternative bases whether derived from natural sources or synthesized.Such hybridizing or complementary nucleic acid, however, is definedfurther as being novel and unobvious over any prior art nucleic acidincluding that which encodes, hybridizes under stringent conditions, oris complementary to nucleic acid encoding a known peptide, according tothe present invention. “Stringent conditions” are those that (1) employlow ionic strength and high temperature for washing, for example, 0.015MNaCl/0.0015M sodium titrate/0.1% SDS at 50° C., or (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0. 1% Ficoll/0.1%polyinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5× SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS. A skilledartisan can readily determine and vary the stringency conditionsappropriately to obtain a clear and detectable hybridization signal.

[0054] As used herein, a nucleic acid molecule is said to be “isolated”when the nucleic acid molecule is substantially separated fromcontaminant nucleic acid encoding other polypeptides from the source ofnucleic acid.

[0055] B. Methods to Identify Integrin Cytoplasmic Signaling Partners

[0056] Another embodiment of the present invention provides methods foruse in isolating and identifying cytoplasmic signaling partners ofintegrins. Specifically, an integrin containing a β subunit which isphosphorylated on a cytoplasmic tyrosine residue, or a fragment thereofcontaining the phosphorylated cytoplasmic tyrosine residue, can be usedto identify signaling partners from cells which express integrins.

[0057] In detail, an integrin containing a β subunit which isphosphorylated on a cytoplasmic tyrosine residue, or a peptidecontaining the phosphorylated cytoplasmic tyrosine of the β subunit, ismixed with an extract or fraction of a cell which expresses an integrinunder conditions which allow the association of a signaling partner withthe phosphorylated integrin or peptide. After mixing, peptides that havebecome associated with a signaling partner are separated from themixture. The signaling partner that bound the phosphorylated integrin orpeptide can then be removed and further analyzed.

[0058] Any integrin which contains a phosphorylated tyrosine in thecytoplasmic domain of the β subunit can be used for identifying andisolating an integrin cytoplasmic signaling partner. These particularlyinclude the β1, β2, β3, β5, β6, β7 and β8 subunits, but other β subunitsare contemplated. These particularly exclude β subunits in which thephosphorylated tyrosine is followed by an isoleucine or leucine in anITAM motif (YXXI/L). For example, the large β4 cytoplasmic domaincontains an ITAM motif and is excluded from the scope of the presentinvention.

[0059] To identify and isolate a signaling partner, the entire intactintegrin heterodimer, containing an a subunit and β subunit, can beused. Alternatively, an isolated β subunit or a fragment of the βsubunit containing the phosphorylated cytoplasmic tyrosine residue canbe used. In a preferred embodiment, a synthetic peptide corresponding tothe cytoplasmic domain of the β subunit is used. Such a peptide can beof any length so long as it contains the phosphorylated cytoplasmictyrosine residue.

[0060] As used herein, a cellular extract refers to a preparation orfraction which is made from a lysed or disrupted cell. The preferredsource of cellular extracts will be cells which naturally expressintegrins. Examples of such cells include, but are not limited toplatelets and leukocytes. The Examples below demonstrate the use ofextracts of platelets to identify signaling partners which bind theGPIIIa subunit.

[0061] A variety of methods can be used to obtain an extract of a cell.Cells can be disrupted using either physical or chemical disruptionmethods. Examples of physical disruption methods include, but are notlimited to, sonication and mechanical shearing. Examples of chemicallysis methods include, but are not limited to, detergent lysis and theenzyme lysis. A skilled artisan can readily adapt methods for preparingcellular extracts in order to obtain extracts for use in the presentmethods. In Example 1, the detergents NP-40 or Brij 96 were used togenerate platelet extracts.

[0062] The cellular extract can be prepared from cells which have beenfreshly isolated from a subject or from cells or cell lines which havebeen cultured. In addition, the extract can be prepared from cells whichare either in a resting state or from cells which have been activated. Avariety of agents can be used to activate a cell. The selection of anactivating agent will be based on the cell type used. For example,thrombin, collagen or ADP can be used to activate platelets while PMAcan be used to activate leukocytes.

[0063] Once an extract of a cell is prepared, the extract is mixed withthe integrin, or peptide/subunit containing the phosphorylatedcytoplasmic tyrosine, under conditions in which association of thephosphorylated subunit with the signaling partner can occur. A varietyof conditions can be used, the most preferred being conditions whichclosely resemble conditions found in the cytoplasm of anintegrin-expressing cell. Features such as osmolarity, pH, temperature,and the concentration of cellular extract used, can be varied tooptimize the association of the integrin with the signaling partner.

[0064] After mixing under appropriate conditions, the integrins orintegrin peptides are separated from the mixture. A variety oftechniques can be utilized to separate the mixture. For example,antibodies specific to the integrin peptide can be used toimmunoprecipitate the integrin and associated signaling partner.Alternatively standard chemical separation techniques such aschromatography and density/sediment centrifugation can be used.

[0065] In the examples below, a biotin moiety is linked to the syntheticpeptide containing the phosphorylated cytoplasmic tyrosine residue.After mixing, the peptide and associated proteins are separated from themixture using avidin or biotin-specific antibodies.

[0066] After removing nonassociated cellular constituents found in theextract, the signaling partner can be dissociated from theintegrin/signaling partner pair using conventional methods. For example,dissociation can be accomplished by altering the salt concentration orpH of the mixture.

[0067] To aid in separating associated integrin/signaling partners pairsfrom the mixed extract, the β subunit peptide can be immobilized on asolid support. For example, a peptide corresponding to the cytoplasmicdomain of β3 integrin can be attached to a nitrocellulose matrix oracrylic beads. Attachment of the integrin peptide to a solid supportaids in separating the peptide/signaling partner pair from otherconstituents found in the extract.

[0068] The identified signaling partners can be either a single proteinor a complex made up of two or more proteins. In the examples below, apeptide containing the GPIIIa cytoplasmic domain was used to purify apreviously uncharacterized 230 kD protein. In addition, this peptide wasused to isolate a signaling complex containing the shc and grb2proteins.

[0069] C. Use of Isolated Signaling Partners

[0070] Once isolated, the integrin signaling partners obtained using theabove described methods, can be used for a variety of purposes. Thesignaling partner can be used to generate antibodies which bind to thesignaling partner using techniques known in the art. Antibodies whichbind an integrin signaling partner can be used to assay integrinsignaling, as a therapeutic agent to modulate a biological orpathological process mediated by integrin signaling, or to purify thesignaling partner. These uses are described in detail below.

[0071] D. Methods to Identify Agents That Block Integrin CytoplasmicSignaling Partner Interactions

[0072] Another embodiment of the present invention provides methods foridentifying agents which reduce or block the association of an integrinwith a cytoplasmic signaling partner. Specifically, an integrin, a βsubunit of an integrin, or a peptide containing the phosphorylatedcytoplasmic tyrosine of a β subunit. is mixed with a cellular extract inthe presence and absence of an agent to be tested. After mixing underconditions which allow association of the integrin or peptide with asignaling partner, the two mixtures are analyzed and compared todetermine if the agent reduced or blocked the association of theintegrin or peptide with the signaling partner. Agents which block orreduce the association of an integrin with a signaling partner will beidentified as decreasing the amount of association present in the samplecontaining the tested agent.

[0073] As used herein, an agent is said to reduce or blockintegrin/cytoplasmic signaling partner association when the presence ofthe agent decreases the extent to which or prevents the signalingpartner from becoming associated with an integrin or integrin peptidefragment containing the phosphorylated cytoplasmic tyrosine residue. Oneclass of agents will reduce or block the association by binding to thesignaling partner while another class of agents will reduce or block theassociation by binding to the cytoplasmic domain of the β integrin.

[0074] The integrin peptide fragment containing the phosphorylatedcytoplasmic tyrosine residue used in this method can either be theentire isolated β subunit, or a fragment of the β subunit which containsthe phosphorylated cytoplasmic tyrosine residues. In some of theexamples that follow, a synthetic peptide spanning the cytoplasmicdomain of GPIIIa is used.

[0075] The signaling partner used in the above assay can either be afully characterized protein or can be a partially characterized proteinwhich has been identified as being present in a cellular extract. Itwill be apparent to one of ordinary skill in the art that so long as thesignaling partner has been characterized by an identifiable property,e.g., molecular weight, the present assay can be used.

[0076] Agents which are assayed in the above method can be randomlyselected or rationally selected or designed. As used herein, an agent issaid to be randomly selected when the agent is chosen randomly withoutconsidering the specific sequences involved in the association of theintegrin with the signaling partner. An example of randomly selectedagents is the use of a chemical library or a peptide combinatoriallibrary.

[0077] As used herein, an agent is said to be rationally selected ordesigned when the agent is chosen on a nonrandom basis which takes intoaccount the sequence of the target site and/or its conformation inconnection with the agent's action. As described above, there are twosites of action for agents which block integrin/signaling partnerinteraction: the phosphorylated cytoplasmic domain of the β subunit orthe signaling partner. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences which make up the contactsites of the integrin/signaling partner pair. For example, a rationallyselected peptide agent can be a peptide whose amino acid sequence isidentical to the phosphorylated cytoplasmic domain of the integrin. Suchan agent will reduce or block the association of the integrin with thesignaling partner by binding to the signaling partner.

[0078] The agents of the present invention can be peptides, smallmolecules, vitamin derivatives, as well as carbohydrates. A skilledartisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention.

[0079] One class of agents of the present invention are peptide agentswhose amino acid sequences were chosen based on the amino acid sequenceof the cytoplasmic domain of the R subunit of a particular integrin.Such a peptide agent can be modified to prevent dephosphorylation of thetyrosine residue using techniques which are well known in the art. Thenomenclature used to describe the peptide agents follows theconventional practice where the N-terminal amino group is assumed to beto the left and the carboxy groups to the right of each amino acidresidue in the peptide. In the amino acid sequences representing agentsof selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although often not specifically shown, willbe understood to be in the form they would assume at physiological pHvalues, unless otherwise specified. Thus, the N-terminal H⁺ ₂ andC-terminal O⁻ at physiological pH are understood to be present thoughnot necessarily specified and shown, either in specific examples or ingeneric formulas. Free functional groups on the side chains of the aminoacid residues can also be modified by amidation, acylation or othersubstitution, which can, for example, change the solubility of thecompounds without affecting their activity.

[0080] In the peptides shown, each gene-encoded residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the amino acid. In the specificpeptides shown in the present application, the L-form of any amino acidresidue having an optical isomer is intended.

[0081] All of the peptide agents of the invention, when an amino acidforms the C-terminus, may be in the form of the pharmaceuticallyacceptable salts or esters. Salts may be, for example, Na⁺, K⁺, Ca⁺²,Mg⁺² and the like; the esters are generally those of alcohols of 1-6C.In all of the peptides of the invention, one or more amide linkages(—CO—NH—) may optionally be replaced with another linkage which is anisostere such as —CH₂NH—, —CH₂S—, —CH₂CH₂, —CH═CH— (cis and trans),—COCH₂—, —CH(OH)CH₂— and —CH₂SO—. This replacement can be made bymethods known in the art. The following references describe preparationof peptide analogs which include these alternative-linking moieties:Spatola, A. F., Vega Data “Peptide Backbone Modifications” (generalreview) (1983) 1(3); Spatola, A. F., in “Chemistry and Biochemistry ofAmino Acids Peptides and Proteins,” B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983) (general review); Morley, J. S., Trends PharmSci (1980) pp. 463468 (general review); Hudson, D, et al., Int J PeptProt Res (1979) 14:177-185 (—CH₂NH—, —CH₂CH₂—); Spatola, A. F., et al.,Life Sci (1986) 38:1243-1249 (—CH₂—S); Hann, M. M., J Chem Soc PerkinTrans I (1982) 307-314 (—CH—CH—, cis and trans); Almquist, R. G., etal., J Med Chem (1980) 23:1392-1398 (—COCH₂—); Jennings-White, C., etal., Tetrahedron Lett (1982) 23:2533 (—COCH₂—); Szelke, M., et al.,European Application EP 45665 (1982) CA:97:39405 (—CH(OH)CH₂—);Holladay, M. W., et al., Tetrahedron Lett (1983) 24:4401-4404(—C(OH)CH₂—); and Hruby, V. J., Life Sci (1982) 31:189-199 (—CH₂—S—).

[0082] The peptide agents of the invention can be prepared usingstandard solid phase (or solution phase) peptide synthesis methods, asis known in the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation and produced recombinantly using standard recombinantproduction systems. The production using solid phase peptide synthesisis necessitated if non-gene-encoded amino acids are to be included.

[0083] Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the phosphorylated cytoplasmicdomain of an integrin or with an integrin signaling partner. Antibodyagents are obtained by immunization of suitable mammalian subjects withpeptides containing as antigenic regions those portions of thephosphorylated cytoplasmic domain or signaling partner intended to betargeted by the antibodies. Critical regions include the contact sitesinvolved in the association of the integrin with a signaling partner.

[0084] Antibody agents are prepared by immunizing suitable mammalianhosts in appropriate immunization protocols using the peptide haptensalone, if they are of sufficient length, or, if desired, or if requiredto enhance immunogenicity, conjugated to suitable carriers. Methods forpreparing immunogenic conjugates with carriers such as BSA, KLH, orother carrier proteins are well known in the art. In some circumstances,direct conjugation using, for example, carbodiimide reagents may beeffective; in other instances linking reagents such as those supplied byPierce Chemical Co., Rockford, Ill., may be desirable to provideaccessibility to the hapten. The hapten peptides can be extended at theamino or carboxy terminus with a Cys residue or interspersed withcysteine residues, for example, to facilitate linking to carrier.Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

[0085] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions, useof monoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the peptide hapten or isthe integrin or signaling partner itself When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in ascitesfluid.

[0086] The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

[0087] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of receptor can also be produced in the context ofchimeras with multiple species origin.

[0088] The antibodies thus produced are useful not only as modulators ofthe association of an integrin with a signaling partner, but are alsouseful in immunoassays for detecting integrin mediated signaling and forthe purification of integrin-associated signaling proteins.

[0089] E. Uses for Agents that Block the Association of an Integrin witha Signaling Partner

[0090] As provided in the Background section, integrins play importantroles in intracellular signaling, cellular attachment, cellularaggregation and cellular migration.

[0091] Agents which reduce or block the interactions of an integrin witha cytoplasmic signaling partner can be used to modulate biological andpathologic processes associated with integrin function and activity.

[0092] In detail, a biological or pathological process mediated by anintegrin can be modulated by administering to a subject an agent whichblocks the interaction of an integrin with a cytoplasmic signalingpartner.

[0093] As used herein, a subject can be any mammal, so long as themammal is in need of modulation of a pathological or biological processmediated by an integrin. The term “mammal” is meant an individualbelonging to the class Mammalia. The invention is particularly useful inthe treatment of human subjects.

[0094] As used herein, biological or pathological process mediated by anintegrin refers to the wide variety of cellular events in which anintegrin binds a substrate producing an intracellular signal. Examplesof biological processes include, but are not limited to, cellularattachment or adhesion to substrates and other cells, cellularaggregation, cellular migration, cell proliferation, and celldifferentiation.

[0095] Pathological processes refers to a category of biologicalprocesses which produce a deleterious effect. For example, thrombosis isthe deleterious attachment and aggregation of platelets while metastasisis the deleterious migration of tumor cells. These pathologicalprocesses can be modulated using agents which reduce or blockintegrin/signaling partner association.

[0096] As used herein, an agent is said to modulate a pathologicalprocess when the agent reduces the degree or severity of the process.For example, an agent is said to modulate thrombosis when the agentreduces the attachment or aggregation of platelets.

[0097] 1. Biological and Pathological Process Mediated by IntegrinsContaining the β1 Subunit:

[0098] A number of different a subunits can combine with the β1 subunit.These integrins form a group of proteins known as VLA (very lateantigens) since their appearance on certain cell types is upregulatedoften hours to days post-activation.

[0099] α1β1 (VLA-1) binds both collagen and laminin and is present on anumber of cell types, including smooth muscle cells, monocytes andactivated T lymphocytes. This integrin plays a role in certain humanintestinal inflammatory diseases (MacDonald, T. T. et al., J Clin Path(1990) 43:313-315).

[0100] α2β1 (VLA-2) also binds collagen and has a widespread cellulardistribution. It is present on both B and T lymphocytes, fibroblasts,endothelial cells and platelets (Giltay, J. C. et al., Blood(1989)73:1235-1241). This integrin plays an important role in wound healing(Schiro, J. A. et al., Cell (1991) 67:403-410).

[0101] α3β1 (VLA-3) binds multiple ligands including laminin andcollagen. Its cellular distribution is also broad and this integrin isinvolved in cell-cell adhesion (Kaufman, R. et al., J Cell Biol (1989)109:1807-1815).

[0102] α4β1 (VLA-4) binds fibronectin and VCAM-1, with its expressionbeing restricted mostly to cells of the immune system.

[0103] α5β1 (VLA-5) also functions as a fibronectin receptor and has awide cellular distribution.

[0104] α6β1 (VLA-6) mediates adhesion to laminin and again is widelydistributed.

[0105] These latter four integrins have been shown to act asco-stimulatory receptors for T lymphocytes which have been partiallyactivated by T cell receptor stimuli, and a number of the β1 integrinshave been shown to have outside-in signaling (reviewed in Hynes, R. O.Cell (1992) 69:11-25).

[0106] 2. Biological and Pathological Processes Mediated by IntegrinsContaining the β2 Subunit:

[0107] The β2 subunit can pair with three different a subunits, namelyαL, αM and αX. All three of these integrins are expressed mainly incells of the immune system and have been shown to play roles ininflammatory processes. Patients exist who lack the β2 subunit (termedLAD, leukocyte adhesion deficiency) and these patients show an increasedsusceptibility to bacterial infections, chronic granulocytosis and lackof pus formation (reviewed in Anderson, D. C. et al., Ann Rev Med (1987)38:175-194).

[0108] αLβ2 (LFA-1, leukocyte function associated antigen 1) is found onlymphocytes, granulocytes, monocytes and macrophages. Its expressionlevels are increased on memory T cells. This integrin functions inmediating the binding of leukocytes to the epithelium duringinflammatory responses, a process that involves its binding to theligand ICAM-1 (intracellular adhesion molecule 1). It is also involvedin a number of the immune functions carried out by T cells, e.g.,adhesion of cytotoxic T cells to their targets (reviewed in Larson, R.S. et al., Immunol Rev (1990) 114:181-217).

[0109] αMβ2 (Mac-1) found on monocytes, macrophages, granulocytes and NKcells. This integrin mediates adherence to both matrix and cell surfaceproteins. Its ligands include fibrinogen, factor X, C3bi and ICAM-1. Itplays a role in neutrophil binding to endothelial cells and subsequentextravasation to sites of inflammation.

[0110] αXβ2 (p150,95) is expressed on monocytes, granulocytes, activatedB and T cells, NK cells and at high levels on macrophages. It is amarker for hairy leukemia cells. Fibrinogen is a ligand for thisintegrin. Again, this integrin appears to be involved in inflammatoryresponses, playing a role in monocyte and granulocyte adhesion toendothelial cells although the ligand on the endothelial cells remainsunidentified (see Larson and Springer review).

[0111] 3. Biological and Pathological Process Mediated by IntegrinsContaining the β3 Subunit:

[0112] Two known parings of the β3 subunit have been observed: with αVto make αVβ3, the Vitronectin Receptor; and with GPIIb to makeGPIIb-IIIa, the Fibrinogen Receptor. αVβ3 is widely distributed, is themost promiscuous member of the integrin family and mediates cellularattachment to a wide spectrum of adhesive proteins, mostly at the R-G-Dsequence on the adhesive protein. The biological processes mediated byαVβ3 are diverse and include bone resorption, angiogenesis, tumormetastasis and restenosis. αVβ3 is known to signal upon adhesive proteinligation (Leavesley, P. I. et al., J. Cell Biol (1993) 121:163-170). Asan example, endothelial cells undergo apoptosis when relieved ofligation (Brooks, P. C. Cell (1994) 79:1157-1164).

[0113] GPIIb-IIIa, by contrast, is restricted to platelets and cells ofmegakaryocyted lineage although a report has appeared indicating thatGPIIb-IIIa is present in tumor cell lineages. As discussed in detailelsewhere in this application, the function of GPIIb-IIIa is primarilyto bind adhesive proteins to mediate platelet aggregation. In thisfunction, GPIIb-IIa participates in both inside-out and outside-insignaling. Decreased receptor function of GPIIb-IIIa leads to bleeding;elevated receptor function of GPIIb-IIIa can lead to thrombus formation.Studies have appeared indicating that platelet aggregation throughGPIIb-IIIa may also be involved in tumor metastasis.

[0114] 4. Biological and Pathological Processes Mediated by IntegrinContaining the β5 Subunit:

[0115] The exact function of this integrin is not clear, howeverevidence exists that activation of a receptor tyrosine kinase isrequired in order for carcinoma cells to migrate on vitronectin usingαvβ5 (Klemke, R. L. et al., J Cell Biol (1994) 127:859-866).Interestingly, this integrin (as well as the αvβ3 integrin) may act asreceptors for adenovirus entry into cells (reviewed in Nemerow, G. R. etal., Trends in Cell Biol (1994) 4:52-55).

[0116] 5. Biological and Pathological Processes Mediated by IntegrinContaining the β6 Subunit:

[0117] β6 is expressed during fetal development, as well as woundhealing and in epithelial tumors which suggests it may play a role inepithelial migration. Its expression can be induced in keratinocytestreated with TGF-β1, implying a role for this receptor in wound healing(Zambruno, G. et al. , J Cell Biol (1995) 129(3):853-865). This integrinbinds to fibronectin and there is evidence that the β6 cytoplasmicdomain is important for some of its function. Expression of αvβ6 in ahuman carcinoma cell line enhances the proliferative ability of thesecells in in vitro and in vivo assays, but mutant with deletions of thecarboxy terminal of the β6 cytoplasmic domain no longer showed thisenhanced proliferation (Agrez, M. et al., J Cell Biol (1994)127:547-556).

[0118] 6. Biological and Pathological Processes Mediated by IntegrinContaining the β7 Subunit:

[0119] Two β7 containing integrins have been identified thus far. Theseare α4β7 and αEβ7 and appear to be expressed mostly on leukocytes, inparticular small intestinal epithelial lymphocytes (Ni, J. et al., CellImmunol (1995) 161:166-172). Their expression can also be induced onperipheral monocytes, and monocytoid cell lines, with agents such as PMAand IFNg, which promote maturation to the macrophage stage ofdevelopment (Tiisala, S. et al., Eur J Immunol (1995) 25:411-417). Theseintegrins play a role in intraepithelial homing. The ligands for α4β7include fibronectin and VCAM-1 but no ligands have yet been identifiedfor αEβ7.

[0120] F. Inhibitors of Kinases that Phosphorylate Integrins

[0121] The present invention further provides methods for reducing orblocking integrin mediated signaling which rely on blocking or reducingthe level of phosphorylation of tyrosine residues found in thecytoplasmic domain of β-subunit. Since tyrosine phosphorylation in vivois required for the association of an integrin with a signaling partner,reduction or blockage of tyrosinyl phosphorylation will result inmodulating, reducing or eliminating integrin mediated signals.

[0122] In detail, integrin mediated signaling can be reduced or blockedby administering to a cell an agent which inhibits the action oftyrosine kinases. Alternatively, signaling can be reduced by supplyingexogenous tyrosine phosphatase or by increasing the activity of cellularphosphatase.

[0123] As used herein, an agent is said to inhibit the action of atyrosine kinase if the agent can reduce or eliminate the activity of atyrosine kinase. A number of agents are presently known which can reducethe activity of tyrosine kinase, these include, but are not limited toGenistein and Herbimycin A (a bezenoid ansamycin inhibitor).

[0124] As used herein, an agent is said to increase the activity of acellular phosphatase if the agent stimulates the production of cellularphosphatases or increases the activity of phosphatases present in acell. Phosphatases can be activated by addition of PK-A or PK-Cactivators or inhibitors of serine/threonine phosphorylation (Brautigan,D. L. et al., PNAS USA (1991) 15:6696).

[0125] G. Administration of Agents that Affect Integrin Signaling

[0126] The agents of the present invention can be provided alone, or incombination with another agents that modulate a particular pathologicalprocess. For example, an agent of the present invention that reducesthrombosis by blocking integrin mediated cellular signaling can beadministered in combination with other anti-thrombotic agents. As usedherein, two agents are said to be administered in combination when thetwo agents are administered simultaneously or are administeredindependently in a fashion such that the agents will act at the sametime.

[0127] The agents of the present invention can be administered viaparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,transdermal, or buccal routes. Alternatively, or concurrently,administration may be by the oral route. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

[0128] The present invention further provides compositions containingone or more agents which block integrin/signaling partner association.While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typical dosages comprise 0.1 to 100 mg/kg/body wt. The preferred dosagescomprise 1 to 100 mg/kg/body wt. The most preferred dosages comprise 10to 100 mg/kg/body wt.

[0129] In addition to the pharmacologically active agent, thecompositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate for oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides. Aqueous injection suspensionsmay contain substances which increase the viscosity of the suspensionand include, for example, sodium carboxymethyl cellulose, sorbitol,and/or dextran. Optionally, the suspension may also contain stabilizers.Liposomes can also be used to encapsulate the agent for delivery intothe cell.

[0130] The pharmaceutical formulation for systemic administrationaccording to the invention may be formulated for enteral, parenteral ortopical administration. Indeed, all three types of formulation may beused simultaneously to achieve systemic administration of the activeingredient.

[0131] Suitable formulations for oral administration include hard orsoft gelatin capsules, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof.

[0132] In practicing the methods of this invention, the compounds ofthis invention may be used alone or in combination, or in combinationwith other therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice, such as anticoagulant agents,thrombolytic agents, or other antithrombotics, including plateletaggregation inhibitors, tissue plasminogen activators, urokinase,prourokinase, streptokinase, heparin, aspirin, or warfarin. Thecompounds of this invention can be utilized in vivo, ordinarily inmammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, ratsand mice, or in vitro.

[0133] The preferred therapeutic compounds of the present invention arecharacterized by their ability to inhibit thrombus formation withacceptable effects on classical measures of coagulation parameters,platelets and platelet function, and acceptable levels of bleedingcomplications associated with their use. Conditions characterized byundesired thrombosis would include those involving the arterial andvenous vasculature.

[0134] With respect to the coronary arterial vasculature, abnormalthrombus formation characterizes the rupture of an establishedatherosclerotic plaque which is the major cause of acute myocardialinfarction and unstable angina, as well as also characterizing theocclusive coronary thrombus formation resulting from either thrombolytictherapy or percutaneous transluminal coronary angioplasty (PTCA).

[0135] With respect to the venous vasculature, abnormal thrombusformation characterizes the condition observed in patients undergoingmajor surgery in the lower extremities or the abdominal area who oftensuffer from thrombus formation in the venous vasculature resulting inreduced blood flow to the affected extremity and a predisposition topulmonary embolism. Abnormal thrombus formation further characterizesdisseminated intravascular coagulopathy which commonly occurs withinboth vascular systems during septic shock, certain viral infections andcancer, and is a condition where there is rapid consumption ofcoagulation factors and systemic coagulation which results in theformation of life-threatening thrombi occurring throughout themicrovasculature leading to widespread organ failure.

[0136] The compounds of this present invention, selected and used asdisclosed herein, or otherwise selected and used by techniques known tothe skilled artisan, are believed to be useful for preventing ortreating a condition characterized by undesired thrombosis, such as in(a) the treatment or prevention of any thrombotically mediated acutecoronary syndrome including myocardial infarction, unstable angina,refractory angina, occlusive coronary thrombus occurringpost-thrombolytic therapy or post-coronary angioplasty, (b) thetreatment or prevention of any thrombotically mediated cerebrovascularsyndrome including embolic stroke, thrombotic stroke or transientischemic attacks, (c) the treatment or prevention of any thromboticsyndrome occurring in the venous system including deep venous thrombosisor pulmonary embolus occurring either spontaneously or in the setting ofmalignancy, surgery or trauma, (d) the treatment or prevention of anycoagulopathy including disseminated intravascular coagulation (includingthe setting of septic shock or other infection, surgery, pregnancy,trauma or malignancy and whether associated with multi-organ failure ornot), thrombotic thrombocytopenic purpura, thromboangiitis obliterans,or thrombotic disease associated with heparin induced thrombocytopenia,(e) the treatment or prevention of thrombotic complications associatedwith extracorporeal circulation (e.g., renal dialysis, cardiopulmonarybypass or other oxygenation procedure or plasmapheresis), (f) thetreatment or prevention of thrombotic complications associated withinstrumentation (e.g., cardiac or other intravascular catheterization,intra-aortic balloon pump, coronary stent or cardiac valve), and (g)those involved with the fitting of prosthetic devices.

[0137] Anticoagulant therapy by agents according to the presentinvention also are useful to prevent coagulation of stored whole bloodand to prevent coagulation in other biological samples for testing orstorage. Thus the compounds of this invention can be added to orcontacted with any medium containing blood cells and in which it isdesired that blood coagulation be inhibited, e.g., when contacting amammal's blood with material such as vascular grafts and stents,orthopedic prostheses, cardiac stents, valves and prostheses,extracorporeal circulation systems and the like.

[0138] H. Methods for Identifying Integrin-Mediated Signaling

[0139] The present invention further provides methods for identifyingcells involved in integrin-mediated signaling as well as techniqueswhich can be applied to diagnose biological and pathological processesassociated with integrin-mediated signaling.

[0140] Specifically, integrin-mediated signaling can be identified bydetermining whether the cytoplasmic domain of the β subunit of anintegrin expressed by a particular cell is phosphorylated. Cellspossessing nonphosphorylated cytoplasmic domains are not considered tobe involved in integrin-mediated signaling while cells possessingphosphorylated cytoplasmic domains are. Such methods are useful inidentifying sites of inflammation, thrombosis, and tumor metastasis.

[0141] In detail, an extract of cells is prepared which contains the Psubunits of the cellular integrins. The cytoplasmic domains are thenassayed to determine whether the tyrosine residues contained in thecytoplasmic domain are phosphorylated. The degree of phosphorylatedtyrosines present in the cytoplasmic domain of the β subunits provides ameasurement of the degree the cell is participating in signaling. Anincrease in the degree of signaling is a measurement of the level ofintegrin mediated activity.

[0142] For example, to determine whether a tumor has metastaticpotential, an extract is made of the tumor cells and the β subunit ofintegrins expressed by the tumor cells are isolated using known methodssuch as immunoprecipitation. The cytoplasmic domain of the β subunits isthen analyzed, for example, by 2-D gel electrophoresis to determine thepresence or absence of phosphorylated tyrosine residues. The presence ofa phosphorylated tyrosine residue correlates with the metastaticpotential of the cancer.

[0143] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLES

[0144] Experimental Procedures—Platelet preparation

[0145] Blood from healthy volunteers was collected into ⅙^(th) volume of85 mM sodium citrate, 111 mM dextrose and 71 mM citric acid supplementedwith 50 ng/ml PGI2 and 0.6 U/ml apyrase. After centrifugation of theblood at 160× g for 20 minutes the platelet rich plasma was removed andsubjected to further centrifugation at 730× g for 10 minutes to sedimentthe platelets. Platelets were then resuspended in 13 mM trisodiumcitrate, 120 mM NaCl and 30 mM dextrose pH 7.0 and washed x 2 with thisbuffer. The platelets were then resuspended in 12 mM NaHCO3, 138 mMNaCl, 5.5 mM dextrose, 2.9 mM KCl, 10 mM Hepes, 1 mM CaCl₂, 0.3 U/mlapyrase and allowed to recover for 1 hour at 37° C. Resting plateletswere activated by the addition of 1 nM thrombin with stirring untilaggregates were visible (1-3 minutes).

[0146] Antibodies

[0147] The anti-phosphotyrosine antibodies, 4G10 and PY-20, wereobtained from UBI (Lake Placid, N.Y.) and Transduction Laboratories(Lexington, Ky.), respectively . The anti-tyrosine kinase antibodiesused in these experiments were as follows: mouse anti-p60c-srcmonoclonal antibody (mAb) 327 (Oncogene Science, Uniondale, N.Y.),rabbit anti-human p53/561yn antisera (UBI), rabbit anti-p59fyn antiseraand rabbit anti-human syk antisera (Santa Cruz Biotechnology, SantaCruz, Calif.). The mouse anti-human GPIIIa monoclonal antibodies E8 andC3a.19.5 were raised against purified GPIIIa and a KLH-coupledcytoplasmic domain peptide of GPIIIa respectively. Rabbit anti-GRB2antisera was from Santa Cruz Biotechnology. Rabbit anti-vav antibody waspurchased from UBI. The rabbit anti-SHC antibody came from TransductionLaboratories.

[0148] In vitro Kinase Assays

[0149] The desired tyrosine kinase, either purified (p60^(c-src),Oncogene Science, or p93^(c-fes), UBI) or partially purified (p56^(lyn),p59^(fyn,) both from UBI) was incubated with the relevant substrate in20 ml final volume kinase assay buffer (20 mM Tris-HCl pH 7.4, 10 mMMgCl₂, 10 mM MnCl₂, 0.1% NP-40) with 5-10 mCi of ³²P-g-ATP (Amersham,Arlington Heights, Ill.) for 10 minutes at room temperature. Thesubstrates were present at the following concentrations: affinitypurified GPIIbIIIa 2.5 mM, enolase 1.5 mM, or cytoplasmic domain peptideof GPIIIa 25 mM. Reactions were terminated by the addition of 20 ml 2×Laemmli sample buffer and boiled for 5 min. prior to loading onto gels.Bands were visualized by autoradiography using Kodak X-OMAT LS film. Forsome experiments the relevant tyrosine kinase was immunoprecipitatedfrom platelet lysates using the specific anti-kinase antibody plusprotein A-sepharose. The beads were then washed twice in lysis buffer,and once in kinase assay buffer prior to performing the kinase reaction.The kinase reaction was terminated by the addition of kinase wash buffer(10 mM Tris-HCl pH 7.2, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100/0.3%SDS) and washed twice with this buffer.

[0150] Beads were then resuspended in 40 ml of Laemmli sample buffer,boiled for 5 min. and the supernatants loaded onto gels. Experimentslooking at kinase activity associated with the GPIIIa protein were alsoperformed as detailed above except that GPIIIa was immunoprecipitatedfrom lysates of platelets (1% Brij 96, 150 mM NaCl, 10 mMtriethanolamine, 1 mM EDTA, 1 mM sodium vanadate, 1 mM PMSF, 20 mMleupeptin, 0.15 U/ml aprotinin) and these immunoprecipitates subjectedto an in vitro kinase assay which was terminated and washed in thekinase wash buffer without detergents present. Co-immunoprecipitationexperiments Control or thrombin-aggregated platelets were lysed in the1% Brij 96 lysis buffer. The detergent-soluble lysates were subjected toimmunoprecipitation with protein A sepharose plus an anti-tyrosinekinase antibody, the anti-GPIIIa monoclonal antibody (E8), or therelevant control antibody (either pre-immune rabbit antisera or aclass-matched mouse IgG). The beads were then washed once in lysisbuffer+0.5M NaCl, and twice in lysis buffer prior to boiling in Laemmlisample buffer Samples were run on 7-9% SDS polyacrylamide gels andtransferred to nitrocellulose. Membranes were blocked in TBS-NP-40/4%BSA (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% NP-40), followed byincubation with the desired antibody. Immunoreactive bands werevisualized by incubating the blots for 1 h in HRP-conjugated anti-mouseor anti-rabbit IgG antibody and employing chemiluminscent detection (ECLdetection kit, Amersham).

[0151] Phosphopeptide Synthesis

[0152] Peptides consisting of cytoplasmic regions of GPIIIa weresynthesized by SynPep corporation using solid phase Fmoc chemistry.Peptides were dissolved in 0.1% TFA/50% acetonitrile and diluted asneeded.

[0153] Peptide Precipitations

[0154] Biotinylated peptides (1-10 mM) were incubated with plateletlysates (either 1% NP-40 or 1% Brij 96 lysis buffer) for 90 min. at 4°C. Avidin-agarose beads were then added for a further 90 min. toprecipitate the biotinylated peptides and any associated proteins. Thebeads were washed and then boiled in Laemmli sample buffer. The proteinswere separated on SDS PAGE gels and transferred to nitrocellulose forimmunoblotting with antibodies to various signaling proteins.

[0155] In vivo Tyrosine Phosphorylation of GPIIIa

[0156] 330 μl of 3× non-reducing sample buffer containing 1 mM sodiumvanadate was added to 1 ml of control or thrombin-aggregated platelets.After boiling, samples corresponding to ˜1-2×10⁸ platelets wereseparated in the first dimension on 7% SDS-PAGE gels. For the seconddimension separation the strips were boiled in reducing sample bufferand then run on a 5% SDS-PAGE gel. These experiments were carried out induplicate and one gel was stained with Coomassie blue to confirm totalprotein loading integrity while the other gel was transferred tonitrocellulose. This membrane was then immunoblotted withanti-phosphotyrosine antibodies and the relative amount ofphosphorylated GPIIIa was determined by densitometry for both controland thrombin-aggregated samples. The blot was then stripped byincubation for 30 min. at 50° C. in 1 00 mM 2-mercaptoethanol, 2% sodiumdodecyl sulfate, 62.5 mM Tris-HCl pH 6.7 and reblocked before reprobingwith the anti-GPIIIa mAb C3a.19.5. Again densitometry was performed toensure equal amounts of GPIIIa were present in both control andthrombin-aggregated samples.

Example 1 Cytoplasmic Tyrosine Phosphorylation Sites on IntegrinSub-Units

[0157] 1. GPIIIa Tyrosine Phosphorylation in Response toThrombin-Stimulated Aggregation

[0158] GPIIb-IIIa is a member of the integrin superfamily of proteinsdescribed above. GPIIb-IIIa is used as the prototype integrin for theinvention described herein. Because of the high degree of sequencehomology between integrin subunits, this invention can be applied to thefamily of integrins described above. Two tyrosine residues are presentin the cytoplasmic domain of GPIIIa. Data presented below demonstratethat i) one of the cytoplasmic tyrosines exists in a sequence withstructural features that predict that the protein should be a tyrosinekinase substrate (e.g., the structural similarity to the tyrosinephosphorylated motif found in the epidermal growth factor and insulinreceptor referred to above) and ii) that the tyrosine phosphorylatedcytoplasmic domain should create a motif(s) which will interact withsignaling proteins.

[0159] In light of our discovery, the following observations arerelevant. The NPLY sequence encompassing residues 744-747 of GPIIIa ishomologous to the NPXY motif which, when phosphorylated on tyrosine, isknown to bind proteins with the phosphotyrosine-binding (PTB) domainsuch as SHC, IRS-1, and possibly pp140 kDa (Kavanaugh, W. M. et al.,Science (1994) 266:1862-1865; Gustafson, T. A. et al., Mol Cell Biol(1995) 15:2500-2508. Also there exists an immune receptor tyrosine-basedactivation motif (ITAM; YXXL/IXXXXXXXXYXXL/I) found on subunits of the Tcell receptor, B cell receptor, and Fc receptor which are, whenphosphorylated on both tyrosines, known to interact with signalingproteins (e.g. ZAP-70 in T cells or syk in B cells) (Chan, A. C. et al.,Cell (1992) 71:649-662; Hutchcroft, J. E. et al., J Biol Chem (1992)267:8613-8619; Law, D. A. et al., Curr Biol (1993) 3:645-657). It isnoted that the sequence in the β3 subunit, although containing twotyrosine residues, lacks the L/I residues found in all ITAM domains.Therefore, the §3 cytoplasmic domain does not appear to contain an ITAMmotif. However, the cytoplasmic domain of the P4 integrin, which doesnot bear homology to the other integrin β subunits, does contain an ITAMdomain. Like other ITAMs, this domain has recently been shown to act inthe recruitment of signaling molecules (Mainiero, F. et al., EMBOJ(1995) 14:4470-4481). Accordingly, experimental protocols weredeveloped to determine whether the tyrosine residues within the GPIIIawere also phosphorylated in response to stimuli which activate theGPIIb-IIIa integrin.

[0160] Resting platelets were induced to aggregate by the addition ofthe agonist thrombin with stirring and lysates were made from bothcontrol and aggregated platelets.

[0161] When GPIIb-IIIa was immunoprecipitated from these lysates, notyrosine phosphorylation of GPIIIa was observed in either sample whenthese immunoprecipitates were analyzed following SDS disc gelelectrophoresis, as has been previously reported (Parise, L. V. et al.,Blood, (1990) 75:2363-2368).

[0162] However, a three to six fold enhancement of phosphorylation ofGPIIIa was observed when platelets were lysed in a high concentration ofSDS and the proteins immediately separated by 2-D gel electrophoresisprior to transfer to a nitrocellulose membrane, and immunoblotting todetect phosphorylated proteins with phosphotyrosine antibodies and witha GPIIIa antibody to quantitate GPIIIa. These results show that theGPIIIa protein is phosphorylated on tyrosines, in vivo, in response tothrombin-induced aggregation (FIG. 3). Although the reason why enhancedphosphorylation of GPIIIa is not observed when lysates areimmunoprecipitated with GPIIb-IIIa antibodies is not known, it ispossible that the high concentration of protein phosphatases present inplatelets (Frangioni, J. V. et al., EMBO J(1993) 12:4843-4856) precludedidentification of the phosphorylated species.

[0163] Additional experiments have been performed using a 2-D gelelectrophoresis protocol and have yielded further information on thetyrosine phosphorylation of GPIIIa (FIG. 3). GPIIIa tyrosinephosphorylation can be observed in platelets induced to aggregate usingagonists other than thrombin. For example, treating platelets with ADPand fibrinogen allows for platelet aggregation and tyrosinephosphorylation of GPIIIa was seen. In all cases platelet aggregationappears to be a prerequisite for GPIIIa tyrosine phosphorylation. Whenplatelets were stimulated with ADP alone, which allows for inside-outactivation of GPIIb-IIIa but does not induce platelet aggregation, noincrease in GPIIIa tyrosine phosphorylation was noted (See Table 1).These results indicate that the tyrosine phosphorylation of GPIIIa ismost likely a consequence of outside-in GPIIb-IIIa signaling. TABLE 1Platelet aggregation appears to be required for GP IIIa (β3) tyrosinephosphorylation. Platelets were stimulated with the indicated agonists.The tyrosine phosphorylation state of β3 was than determined using 2-Dgel electrophoresis and anti-phosphotyrosine immunoblotting. AgonistAggregation β3 Phosphorylation Thrombin no no Thrombin YES YES ADP no noADP, fibrinogen no no ADP, fibrinogen YES YES

[0164] A number of cytoplasmic tyrosine kinases have been reported to bepresent in platelets and indeed several have been implicated in integrinsignaling (Shattil, S. J. et al., Curr Opin Cell Biol (1991) 3:869-879;Clark, E. A. et al., Science, (1995) 268:233-239). This includes thesrc-family tyrosine kinases, src and fyn as well as the non-src-familytyrosine kinase syk. These tyrosine kinases appeared to be possiblecandidates for the kinase responsible for phosphorylating GPIIIa invivo.

[0165] To determine which tyrosine kinases are responsible forphosphorylating GPIIIa in vivo, in vitro kinase reactions were performedto test the ability of a number of these kinases to phosphorylate bothaffinity-purified GPIIb-IIIa as well as a peptide consisting of thecytoplasmic domain of GPIIIa only. Purified, or partially purified,src-family tyrosine kinases p60^(src), p59^(fyn) and p56^(lyn) were allcapable of phosphorylating both the full length GPIIIa and thecytoplasmic domain peptide. The tyrosine kinase syk could alsophosphorylate GPIIIa, although to a lesser degree. In contrast, thetyrosine kinase p93^(fes), which contains an SH2 domain and has beenimplicated in signaling via certain cytokine receptors (Izuhara, K. etal., J Biol Chem (1994) 269:18623-18629), was unable to phosphorylateGPIIIa to any great extent (FIG. 4A). Similar results were obtained whenthe above-mentioned src-kinases and syk were immunoprecipitated fromplatelet lysates and these immunoprecipitates used in in vitro kinaseassays. In these assays the src-family tyrosine kinase p59^(hck) wasalso shown to have the ability to phosphorylate GPIIIa. These dataindicate that members of the src-family of tyrosine kinases, as well asthe syk tyrosine kinase, phosphorylate the cytoplasmic domain of GPIIIain vitro.

[0166] To determine which kinases are associated with GPIIIa inplatelets, both control and thrombin-aggregated platelets were lysed in1% Brij-96, and GPIIb-IIIa was immunoprecipitated and theimmunoprecipitate analyzed for the presence of platelet kinases. Brij-96is a relatively mild detergent yet it appears to solubilize largecomplexes well (Burg, D. L. et al., J Biol Chem (1994) 269:28136-28142).In vitro kinase reactions were performed on the resultantimmunoprecipitates to detect the kinases. An increase in the number of³²P-labeled proteins was observed in the anti-GPIIIa immunoprecipitatesfrom the aggregated as compared to control platelet lysates (FIG. 4B).This suggested that more kinase(s) or more active kinase(s) wasassociating, and therefore co-immunoprecipitating, with the GPIIIa fromthe aggregated platelets. Interestingly, a phosphorylated protein ofapparent molecular weight ˜72 kD was observed in the precipitates fromaggregated cell lysate. This is the same molecular weight of the syktyrosine kinase known to have enhanced tyrosine phosphorylation inresponse to both inside-out and outside-in integrin signal transduction.

[0167] Various kinase re-immunoprecipitation experiments were performedin an attempt to identify the tyrosine kinases associated withGPIIb-IIIa on aggregated platelets. First, GPIIIa was immunoprecipitatedfrom Brij 96 platelet lysates and the immunoprecipitating material waswashed under mild conditions (no detergent) in an attempt to maintainthe integrity of any complexes formed between GPIIIa and cytoplasmictyrosine kinases. The precipitated material was subjected to an in vitrokinase assay followed by re-immunoprecipitation with a variety ofanti-tyrosine kinase antibodies in order to determine whether anykinases could be co-immunoprecipitated with the GPIIIa protein. Toconfirm these results the reverse experiment was also performed wherebyvarious tyrosine kinases were immunoprecipitated from platelet lysatesand, after an in vitro kinase reaction, were subjected tore-immunoprecipitation with an anti-GPIIIa antibody to show associationof the GPIIIa with different tyrosine kinases. However, using theseexperimental protocols no specific tyrosine kinase could be shown toassociate directly with GPIIIa, thus the responsible tyrosine kinase(s)remains unknown.

[0168] The finding that GPIIIa is tyrosine phosphorylated duringplatelet aggregation and identifying the kinases responsible is offundamental importance to understanding the mechanism responsible forintegrin signal transduction which allows for the integrin interactionwith cytoplasmic signaling proteins. Previously, only phosphorylation ofserine on GPIIIa from activated platelets has been observed. No functionof serine phosphorylation is known. In contrast, the tyrosinephosphorylation of a number of cell surface receptor proteins has beenshown to be critical for the signaling ability of these receptors(Weiss, A. et al., Cell (1994) 76:263-274).

[0169] In the case of receptor tyrosine kinases such as the PDGFreceptor, ligand occupancy results in a transphosphorylation of thePDGFR cytoplasmic domains on numerous tyrosine residues. Thephosphorylated tyrosines then act as binding sites for variousSH2-containing proteins, for example shc, src and PLC-g, which areinvolved in effecting the signaling cascade. Other proteins present incell surface immune-receptor complexes also must be phosphorylated ontyrosine residues in order for successful signaling to be initiated.Phosphorylation of the ITAM domains of these receptor complexes recruitsvarious SH2-containing proteins, including tyrosine kinases, to thereceptor complex and initiates a cascade of signaling events. Similarly,tyrosine phosphorylation of GPIIIa can be expected to allow for theinteraction of GPIIb-IIa with signaling proteins; proof that this occursis provided below. The high degree of homology in the domains of GPIIIawhich contain tyrosine to those which exist in other integrins predictsthat the phosphorylation mechanism of the cytoplasmic domain of theintegrin during integrin signal transduction is widely utilized withinthe integrin family of receptors in many different cell types.

Example 2 Interaction of Phosphorylated GPIIIa with CytoplasmicSignaling Proteins

[0170] The discovery that the cytoplasmic domain of GPIIIa isphosphorylated at tyrosine residues during platelet aggregation was thefirst step in demonstrating that the phosphorylated cytoplasmic domainhas functional activity in interacting with signaling proteins. Aphosphorylated peptide corresponding residues 740-762 of GPIIIa wassynthesized and coupled to biotin at the amino terminus: (Peptide1)  Biotin-D-T-A-N-N-P-L-Y(PO₃)-K-E-A-T-S-T-F-T-N-I-T-Y(PO₃)-R-G-T-COOHA control peptide was synthesized with an identical sequence, butunphosphorylated: (Peptide2)  Biotin-D-T-A-N-N-P-L-Y-K-E-A-T-S-T-F-T-N-l-T-Y-R-G-T-COOH

[0171] The phosphorylated peptide was tested to determine whether it hadactivity in interacting with signaling proteins. One method used forthis purpose was adapted from a method used to examine the interactionof the phosphorylated ITAM domain from the T cell receptor withintracellular signaling proteins (Iwashima, M. et al., Science (1994)263:1136-1139). Platelets were lysed with NP-40 detergent, mixed witheither peptide, and the peptide with bound protein isolated byadsorption to avidin-agarose. Shc and Grb2 were two of the proteinswhich specifically bound to the phosphorylated peptide (Peptide 1) (FIG.5).

[0172] The Shc signaling protein also binds to a peptide which wasphosphorylated only on the carboxy-terminal tyrosine residue. To examinethe physiological importance of Grb2 or Shc binding to phosphorylatedGPIIIa, a doubly phosphorylated peptide which was mutated such that theserine at position 752 was changed to a proline was used. This mutationoccurs naturally in a patient with Glanzmann's thrombasthenia and isknown to cause signaling defects (Chen, Y. -P., et al., Blood(1994)84:1857-1865). This peptide was unable to bind Grb2 and showed amarkedly decreased ability to bind Shc. These results suggest that theserine to proline mutation in these patients alters protein structuresuch that interactions with signaling proteins cannot occur.

[0173] Another method was an adaptation of the ligand blot procedureusing the phosphorylated GPIIIa cytoplasmic domain. Total plateletproteins were separated by SDS gel electrophoresis, transferred tonitrocellulose, renatured and incubated with the biotinylated peptides.Several platelet proteins were observed to bind peptide 1 but notpeptide 2, the most prominent of which was a MW=230 kDa protein ofunknown identity. The data show that phosphorylated GPIIIa is capable ofinteracting with many platelet proteins, including a signaling complexwhich contains Shc and Grb2, while the unphosphorylated GPIIIa is not.Since the phosphorylated peptide has this activity, the phosphorylatedpeptide can be used to inhibit GPIIb-IIIa signal transduction throughits ability to compete for signaling proteins. A membrane permeable formof the phosphorylated peptide will therefore have utility as anantithrombotic agent to prevent platelet thrombosis.

[0174] The integrin αVβ3 uses the same β chain (GPIIIa) as doesGPIIb-IIIa. As such, this integrin is phosphorylated during signaltransduction. Therefore, the phosphorylated P chain peptide will haveutility in regulating the signal transduction processes of αVβ3 such asangiogenesis, smooth muscle proliferation and osteoblast boneresorption, and will be useful in the treatment of cancer, restenosisand osteoporosis, respectively.

[0175] As indicated in the Background section and shown in FIG. 2, thecytoplasmic domain of GPIIIa is homologous to that of β1, β2, β5, β6 andβ7. Because the cytoplasmic domains of these β chains all containtyrosines at positions similar to that which is found in GPIIIa (notethat β2 only contains one), and the src family of kinases are widelydistributed, these other integrins are phosphorylated in response tointegrin signal transduction. Although phosphorylation has not beenobserved for any integrin except in vsrc transformed cells (seebackground), it seems probable that the same problems for detectionexist as occurred with detection of GPIIIa phosphorylation in platelets.Note that mutations at the tyrosine-containing region of β1 affected itsfunction (see background). Phosphorylated peptides corresponding to thehomologous sequences in β1, β2, β5, β6 and β7, or mimics of thesesequences, provide the structural basis for agents or targets for agentswhich can be used to inhibit integrin signal transductions in cellscontaining these β subunits. Such agents will have utility for thetreatment of, for example, cancer, restenosis and other diseasesdependent upon excess cell proliferation, inflammatory diseases,Alzheimer's disease, viral infectivity, and atherosclerosis.

Example 3 Identification of Integrin Signaling Proteins

[0176] The discovery that GPIIIa is phosphorylated during plateletaggregation and specifically binds signaling proteins from plateletsallows for the development of methods to discover cytoplasmic signalingproteins whose interaction with integrins is regulated by tyrosinephosphorylation. Phosphorylation on tyrosine may allow for the increasedbinding of proteins with either SH-2 domains or PTB domains, andproteins that bind to phosphorylated integrins should be detectedbecause of their increased binding affinity. Phosphorylated cytoplasmicdomains of the β chains of integrin can be used as probes to probe DNAexpression libraries to detect phosphorylated integrin binding proteins.Method? for such screening have been used to identify proteins that bindto other phosphorylated receptors (Margolis, B., Proc Natl Acad Sci USA(1992) 89:8894-8898; Skolnik, E. Y. et al., Science (1993)260:1953-1955).

[0177] Alternatively, the cytoplasmic domain of GPIIIa can be used as a“bait” in a two hybrid screen in yeast, coexpressing vsrc tophosphorylate the GPIIIa moiety on the “bait”. Proteins whichspecifically bind to the phosphorylated integrin can be identified. Itis recognized that the constitutively active form of csrc may berequired for this purpose. The two hybrid assay has previously been usedto demonstrate the interaction of a phosphorylated protein with asignaling protein, but only on receptor kinases which autophosphorylatesa tyrosine on the same expressed protein fragment.

[0178] This invention provides the method for the identification ofphosphopeptide signaling proteins not associated with andautophosphorylated by a kinase. Proteins identified by these methods canbe expressed and tested for their interactions with phosphorylatedcytoplasmic domains of integrins. Dominant negative proteins, DNAsencoding these proteins, antibodies to these signaling proteins, peptidefragments of these proteins or mimics of these proteins may beintroduced into cells and tested for their abilities to affect integrinfunction and/or used as pharmacological agents to affect integrin signaltransduction.

[0179] Identification of the integrin signaling proteins provides thebasis for a new class of therapeutics which disrupt integrin signaltransduction. The newly identified proteins provide targets forscreening of synthetic small molecule, combinatorial or naturallyoccurring compound libraries to discover agents which affect integrinsignal transduction. These agent provide the basis for a new class oftherapeutics which can be use to regulate integrin function.

[0180] As mentioned above, integrins comprise a family of proteinsinvolved in many biological responses in a variety of cell types. Theresults with the GPIIb-IIIa integrin can be extrapolated to a number ofother integrin family members as the cytoplasmic regions of thedifferent β subunits, in particular β1, β5 and β6, show remarkableconservation of the tyrosine residues and some of the surrounding aminoacids (see FIG. 2).

Example 4 β1-Containing Integrins

[0181] i. In vivo Phosphorylation of β1 Integrins.

[0182] Due to the relatively large numbers of β1-containing integrins, anumber of cell types can be stimulated with the relevant ligands inorder to assess the phosphorylation state of β1 paired to a number of asubunits. For example, α3β1 can be clustered using antibodies in the KBcarcinoma cell line (Kornberg, L. et al., Proc Natl Acad Sci USA (1991)88:8392-8396), or fibronectin can be used as a ligand for the α4β1 andα5β1 integrins on T cells (Yamada, A. et al., J Immunol (1991)146:53-56). Based on observations with the GPIIIa molecule, whereincreases in tyrosine phosphorylation were not seen using traditionalimmunoprecipitation and 1-D gel methods, the 2-D gel technique describedabove is preferred for determining tyrosine phosphorylation.

[0183] ii. Ability of β1 Cytoplasmic Domain Phosphopeptides to Bind toSignaling Proteins.

[0184] The following peptides are used to demonstrate the binding ofsignaling partners to integrins containing a phosphorylated β1 Subunit:(peptide1):  biotin-D-T-G-E-N-P-I-Y(PO₃)-K-S-A-V-T-T-V-V-N-P-K-Y(PO₃)-E-G-K-COOHand the unphosphorylated control peptide (peptide2):  biotin-D-T-G-E-N-P-I-Y-K-S-A-V-T-T-V-V-N-P-K-Y-E-G-K-COOH

[0185] These peptides are incubated with lysates from different cellsincluding platelets and lymphocytes (as well as other cell types inwhich these integrins are expressed). Then avidin-agarose is used toprecipitate any proteins associating with the peptides (experimentalmethod as detailed for GPIIb-IIIa peptides). Proteins associating withthe phosphorylated peptide are signaling proteins and thus targets fortherapeutics. Similarly, the peptides can be used as probes for celllysates as well as cDNA libraries (again detailed above). The ability ofthe phosphopeptide to bind to signaling proteins may also allow it tofunction in a dominant-negative fashion.

[0186] iii. Utilizing Phosphopeptides as Inhibitors of Signaling.

[0187] The ability of the β1 cytoplasmic domain phosphopeptide to bindsignaling proteins indicates that they may act in a dominant-negativefashion when introduced into cells. The antagonistic activity peptidecontaining non-hydrolyzable phosphotyrosyl derivatives can bedemonstrated by permeabilising cells and introducing the peptides.

[0188] The wide cellular distribution, and the number, of β1-containingintegrins allows the use of a variety of cell types for which thereexist well-documented methods of permeabilization. For example, Tlymphocytes can be permeabilised using tetanolysin (Conti, A. et al., JBiol Chem (1993) 268:783-791), monocytes and other immune cells usingstreptolysin-O (Cunha-Melo, J. R., J Immunol (1989) 143:2617-2625) andplatelets using a buffer containing 0.4% n-octyl-b-glucopyranoside(Hartwig, J. H. et al., Cell, (1995) 82:643-653). These methods ofpermeabilising cells result in pore formation that will readily allowfor the peptides described below (approx. 3-5000 kD) to pass into thecells.

[0189] The synthesis of a suitable non-hydrolysable phosphotyrosinepeptide has been described (Burke, T. R. Jr. et al., Tetrahedron Lett(1993) 34:4125-4128; Smyth, M. S. et al., Tetrahedron Lett(1994)35:551-553; and Otaka et al., Tetrahedron Lett(1993)34:7039-7042). In brief, the phosphotyrosine residues are replaced witha difluorophosphonomethyl phenylalanyl group (F₂Pmp) which is resistantto phosphatases. Such peptides, corresponding to the ITAM region of CD3zhave been successfully introduced into permeabilised T cells and showinhibitory function (Wange, R. L. et al., J Biol Chem (1995)270:944-948).

[0190] The effect of introducing the phosphopeptides into permeabilisedcells is assayed in a manner suitable for the cell type used and theparticular β1 integrin present on these cells. For example, the effectof the phosphopeptides on VLA-3 function in fibroblasts can bedetermined using binding to collagen as a readout.

[0191] By performing the experiments mentioned above, targets fortherapeutics designed to inhibit signaling through the β1 integrinfamily can be randomly selected or rationally designed. As is obviousfrom the widespread distribution of these integrins, plus theirinvolvement in a wide range of cellular activities, preventing theinitial signaling event has profound effects on the cellular targets.

Example 5 β5-Containing Integrin

[0192] The β5 integrin subunit contains three tyrosine residues withinits cytoplasmic domain. Residues 774 and 794 have surrounding amino acidsequences similar to those seen in the β3 subunit. Although the spacingseparating the two tyrosine residues is greater (20 amino acids comparedwith 11 for the β3 protein) the two tyrosine residues would be onsimilar faces of an alpha-helix which is a possible structure of the βintegrin cytoplasmic tails. This integrin can pair with the αV subunitto form a vitronectin binding receptor which is expressed onfibroblasts, carcinomas, and hepatoma cells (Diamond, M. S. et al., CurrBiol (1994) 4:506-517).

[0193] A similar set of experiments as those outlined above, for theβ1-containing integrins, are utilized to determine the role of tyrosinephosphorylation in the function of this integrin. Particular detailsapplying to this integrin type are provided below.

[0194] i. In vivo Phosphorylation of β5 Integrin.

[0195] Carcinoma cells, for example the melanoma line M21 or the FGpancreatic carcinoma line, are used in these experiments and activatedby adherence to vitronectin, or possibly by clustering of the integrinusing a specific antibody (P3G2, Wayner, E. A. et al., J Cell Biol(1991) 113:919-929). Cells are then lysed and subjected to 2-D gelelectrophoresis and anti-phosphotyrosine immunoblotting as detailedpreviously.

[0196] ii. Ability of β5 Cytoplasmic Domain Phosphopeptides to Bind toSignaling Proteins.

[0197] The following peptides are used to demonstrate the binding ofsignaling partners to integrins containing a phosphorylated β5 subunit:(peptide1)  biotin-E-M-A-S-N-P-L-Y(PO₃)-R-K-P-I-S-T-H-T-V-D-F-T-F-N-K-F-N-K-S-Y(PO₃)-N-G-T-V-D-COOHAnd the unphosphorylated control peptide: (peptide2)  biotin-E-M-A-S-N-P-L-Y-R-K-P-L-S-T-H-T-V-D-F-T-F-N-K-F-N-K-S-Y-N-G-T-V-D-COOH

[0198] Using the above β5 peptides it was shown that the signalingprotein Shc could bind to the phosphorylated peptide (peptide 1) but notthe unphosphorylated peptide 2. In these experiments the associatedproteins were precipitated from lysates obtained from primary culturesof human umbilical cord venous endothelial cells (HUVECs). These cellsexpress the vitronectin-binding αVβ5 integrin. The results with the β5peptides indicate that extrapolating the results obtained from the P3(GPIIIa) integrin subunit to other integrin β subunits is indeed valid.

[0199] Recent studies have established that tumor cell-inducedangiogenesis can be mediated both by αVβ3, e.g., when angiogenesis isinduced by basic fibroblast growth factor or tissue necrosis factor a,or αVβ5, e.g., when angiogenesis is induced by vascular endothelial cellgrowth factor, transforming growth factor a or by a phorbol ester(Friedlander, M. F. et al., Science (1995) 270:1500-1502). Sincecompetitive antagonists of the αV integrins inhibit tumor growth, thisfinding indicates that inhibitors of αV-integrin signaling will be aneffective anticancer drug. Both αV-containing integrins were alsoexpressed in the actively proliferating vascular cells associated withproliferative diabetic retinopathy, while only αVβ3 was expressed in theactively proliferating vascular cells associated with age-relatedmacular degeneration or presumed ocular histoplasmosis (Friedlander, M.F. et al., Proc Natl Acad Sci USA (1996) 93:9764-9769). Sincecompetitive antagonists of ligand binding to αV-containing integrinsinhibited neovascularization in experimental animals (ibid), thisfinding indicates that inhibitors of αV-containing integrin signaltransduction will also be effective for the treatment of diabeticretinopathy.

[0200] These peptides are used as described above for the β1phosphopeptides, except that associated proteins are precipitated fromsuitable carcinoma cell lysates.

[0201] iii. β5 phosphopeptides as inhibitors of signaling.

[0202] Again, non-hydrolyzable phosphopeptides (containing F₂Pmp groupsas detailed above) are introduced into permeabilized carcinoma cells.The readout for effect on function for this integrin involves bindingand motility on vitronectin.

Example 6 β6-Containing Integrins

[0203] Heterodimers consisting of the αV and β6 subunits have been foundin certain carcinoma cell lines (Busk, M. et al., J Biol Chem (1992)267:5790-5796), as well as epithelial cells (Breuss, J. M. et al., JHistochem Cytochem (1993)41:1521-1527). β6 is expressed during fetaldevelopment, as well as wound healing and in epithelial tumors whichsuggests it may play a role in epithelial migration. Indeed, itsexpression can be induced in keratinocytes treated with TGF-β1 whichimplies a role for this receptor in wound healing (Zambruno, G. et al.,J Cell Biol (1995) 129(3):853-865). This integrin binds to fibronectinand there is evidence that the β6 cytoplasmic domain is important forsome of its function. Expression of αVβ6 in a human carcinoma cell lineenhances the proliferative ability of these cells in in vitro and invivo assays, but mutants with deletions of the carboxy terminal of theβ6 cytoplasmic domain no longer showed this enhanced proliferation(Agrez, M. et al., J Cell Biol (1994) 127:547-556).

[0204] i. In vivo Phosphorylation of β6-Containing Integrin.

[0205] For these experiments a cell line which expresses the αVβ6integrin (e.g. FG-2 carcinoma line) is used. The cells are activatedusing some form of receptor clustering, either with fibronectin or aspecific antibody and then subjected to the 2-D gel andanti-phosphotyrosine immunoblotting procedure previously described.

[0206] ii. Interaction of β6 Phosphopeptides with Signaling Proteins.

[0207] The following peptides are used to demonstrate the binding ofsignaling partners to integrins containing a phosphorylated β6 subunit:(peptide1):  biotin-Q-T-G-T-N-P-L-Y(PO₃)-R-G-S-T-S-T-F-K-N-V-T-Y(PO₃)-K-H-R-E-K-Q-K-V-D-L-S-T-D-C-COOHThe unphosphorylated control peptide: (peptide2):  biotin-Q-T-G-T-N-P-L-Y-R-G-S-T-S-T-F-K-N-V-T-Y-K-H-R-E-K-Q-K-V-D-L-S-T-D-C-COOH

[0208] Alternatively, a phosphorylated peptide missing the 11 carboxyterminal amino acids, which may have an influence on signaling throughthis integrin, can be used.

[0209] This peptide is used to identify signaling proteins which do notrecognize the entire cytoplasmic domain. (peptide3):  biotin-Q-T-G-T-N-P-L-Y(PO₃)-R-G-S-T-S-T-F-K-N-V-T-Y(PO₃)-K-H-R-COOH

[0210] iii. β6 Phosphopeptides as Inhibitors of Signaling.

[0211] The effect of introducing non-hydrolysable derivatives (method asdetailed previously) of the above peptides into permeabilised carcinomaor epithelial cell lines is assessed by cell adhesion to, and migrationon, fibronectin. The β1, β5 and β6 integrin subunits described abovebear the closest similarity to the β3 subunit cytoplasmic domain in thatthey contain at least two tyrosine residues in fairly similar positions.The β2 and β7 subunits have 1 and 2 tyrosines, respectively, in theircytoplasmic domains but in a more membrane proximal position than thoseof the P3 subunit. However, there is high sequence conservation in theamino acids surrounding the 5′ tyrosine in both the β2 and β7 proteinsand these tyrosine residues are found in the same position in each case.These residues, accordingly, are considered to be important. Similarly,the second tyrosine residue present in the β7 cytoplasmic domain is partof an NPXY motif that is found in the β1, β3, β5 and β6 subunits. Thismotif is also present in other signaling proteins and has beenimplicated as having a role in associating with signaling proteins onceits tyrosine is phosphorylated (Gustafson, T. A. et al., Mol Cell Biol(1995) 15:2500-2508; Songyang, Z. et al., J Biol Chem (1995)270:14863-14866).

Example 7 β2-Containing Integrins

[0212] The β2 subunit can pair with three different a subunits, namelyαL, αM and βX. All three of these integrins are expressed mainly incells of the immune system and have been shown to play roles ininflammatory processes. Patients who lack the β2 subunit have leukocyteadhesion deficiency, termed LAD, and these patients show an increasedsusceptibility to bacterial infections, chronic granulocytosis and lackof pus formation (reviewed in Anderson, D. C. et al., Ann Rev Med (1987)38:175-194).

[0213] Experiments have suggested that the β2 subunit has signaltransduction ability. For example, in a COS cell expression system, β2was phosphorylated on serine in response to phorbol ester stimulation.Furthermore, deletion of the β2 cytoplasmic domain inhibited binding ofthe integrin to its ligand (Hibbs, M. L. et al., J Exp Med (1991)174:1227-1238), indicating the functional importance of this region.However, based on results of mutational analysis, these authorsconcluded that phosphorylation of the tyrosine residue in β2 was oflittle or no importance in their assays. Other workers have demonstrateda role for β2 integrins in triggering tyrosine phosphorylation inneutrophils. Berton, G. et al. (J Cell Biol (1994) 126:1111-1121) haveshown that clustering the β2-containing integrin on neutrophils with ananti-β2 antibody, leads to the tyrosine phosphorylation and activationof the src-family tyrosine kinase p58^(fgr).

[0214] αL β2 (LFA-1, leukoctye function associated antigen 1) is foundon lymphocytes, granulocytes, monocytes and macrophages. Its expressionlevels are increased on memory T cells. This integrin functions inmediating the binding of leukocytes to the epithelium duringinflammatory responses, a process that involves its binding to theligand ICAM-1 (intracellular adhesion molecule 1). It is also involvedin a number of the immune functions carried out by T cells, e.g.adhesion of cytotoxic T cells to their targets (reviewed in Larson, R.S. et al., Immunol Rev (1990) 114:181-217).

[0215] αMβ2 (Mac-1) found on monocytes, macrophages, granulocytes and NKcells. This integrin mediates adherence to both matrix and cell surfaceproteins. Its ligands include fibrinogen, factor X, C3bi and ICAM-1. Itplays a role in neutrophil binding to endothelial cells and subsequentextravasation to sites of inflammation (see Larson and Springer review).

[0216] αXβ2 (p150,95) is expressed on monocytes, granulocytes, activatedB and T cells, NK cells and at high levels on macrophages. It is amarker for hairy leukemia cells. Fibrinogen is a ligand for thisintegrin. Again this integrin appears to be involved in inflammatoryresponses, playing a role in monocyte and granulocyte adhesion toendothelial cells although the ligand on the endothelial cells remainsunidentified (see Larson and Springer review). Obviously, all of theP2-containing integrins play an important role in inflammatory andimmune responses.

[0217] i. In vivo Phosphorylation of β2 Integrins.

[0218] Using the methods described above the β2 subunit is shown to havea phosphorylated tyrosine upon integrin activation. These experimentsare carried out in human blood neutrophils, or suitable macrophage orlymphocyte cell lines, depending on the particular β2 integrin studied.The integrins can be activated using immobilized anti-β2 antibody (asdetailed in Berton, G. et al., J Cell Biol (1994) 126:1111-1121) orusing a suitable ligand (e.g. fibrinogen for αXβ2).

[0219] ii. Binding of β2 Cytoplasmic Domain Phosphopeptide to SignalingMolecules.

[0220] The following peptides are used to demonstrate the binding ofsignaling partners to integrins containing a phosphorylated β2 subunit:(peptide1):  biotin-D-L-R-E-Y(PO₃)-R-R-F-E-K-E-K-L-S-Q-W-N-N-D-N-P-L-F-K-S-A-T-COOHAnd the unphosphorylated control peptide:              biotin-D-L-R-E-Y-R-R-F-E-K-E-K-L-S-Q-W-N-N-D-N-P-L-F-K-S-A-T-COOH

[0221] These peptides are used to precipitate any signaling proteins, ingranulocyte or lymphocyte lysates, that bound specifically to thephosphopeptide (as described above for the other β integrinsubfamilies). These peptides are also used to screen leukocyte cDNAlibraries to identify proteins which interact with either thenon-phosphorylated and/or the phosphorylated peptide.

[0222] iii. β2 Phosphopetides as Inhibitors of Signaling.

[0223] The non-hydrolyzable form of the above peptide 1 is introducedinto permeabilised neutrophils or lymphocytes as detailed previously.Assays to test the effect of this phosphopeptide on integrin functioninclude binding to immobilized ligand (ICAM-1 for αLβ2 and αMβ2 andfibrinogen for αXβ2) and the induction of tyrosine phosphorylation afteranti-β2 antibody crosslinking.

Example 8 P7-Containing Integrins

[0224] Two β7-containing integrins have been identified thus far. Theseare α4β7 and αEβ7 and appear to be expressed mostly on leukocytes, inparticular small intestinal epithelial lymphocytes (Ni, J. et al., CellImmunol (1995) 161:166-172). Their expression can also be induced onperipheral monocytes, and monocytoid cell lines, with agents such as PMAand IFNg, which promote maturation to the macrophage stage ofdevelopment (Tiisala, S. et al., Eur J Immunol (1995) 25:411417). Thisintegrin plays a role in intraepithelial homing. The ligands for α4β7include fibronectin and VCAM-1 but no ligands have yet been identifiedfor αEβ7. The β7 subunit contains two tyrosine residues. The membraneproximal tyrosine is conserved between the β2 and β7 subunits and thecarboxy tyrosine is part of the NPXY motif described above.

[0225] i. In vivo Phosphorylation of β7 Integrin.

[0226] Monocytoid lines such as THP-1 or HL-60 are cultured underconditions that induce them to differentiate into macrophage-like cellswhich then express the β7 integrins (Tiisala, S. et al., Eur J Immunol(1995) 25:411-417). The phosphorylation state of the β7 integrin isexamined after stimulation by clustering the integrin with antibody orligand (using the 2-D gel procedure already detailed).

[0227] ii. Ability of β7 Phosphopetides to Bind to Signaling Proteins.

[0228] The following peptides are used to identify signaling proteinsassociated with the β7 cytoplasmic tail in a phospho-dependent manner.These peptides are used to precipitate proteins from suitable celllysates (e.g. differentiated THP-1 cells as described above), and forcDNA library screening.              biotin-D-R-R-E-Y(PO₃)-S-R-F-E-K-E-Q-Q-Q-L-N-W-K-Q-D-S-N-P-L-Y(PO₃)-K-S-A-I-COOHAnd the unphosphorylated control peptide:              biotin-D-R-R-E-Y-S-R-F-E-K-E-Q-Q-Q-L-N-W-K-Q-D-S.N-P-L-Y-K-S-A-I-COOH

[0229] iii. Effect of β7 Cytoplasmic Phosphopetides on Signaling.

[0230] The non-hydrolyzable derivative of the phosphopetide 1 above isintroduced into permeabilised cells (of the type described above). Theeffect of the phosphopeptide on signaling is assessed by assays such asbinding to VCAM-1 or fibronectin.

[0231] As can be seen from the above, the discovery of a mode ofsignaling via GPIIIa tyrosine phosphorylation and the direct binding ofsignaling proteins to the integrin cytoplasmic domain has far-reachingimplications for the signaling and function of other integrin familymembers. The β subunits of the integrins show fairly high levels ofsequence homology, and this is especially true with regards to thetyrosine residues in βs 3, 1, 5 and 6 and to a lesser extent with βs 7and 2. The ability to influence signaling via these integrins usingphosphopetide sequences from the relevant P-integrin cytoplasmic domainswill have profound effects on a wide range of cellular activities.Potential therapeutics arising from such intervention in signaling viathe β-containing integrins are discussed above.

[0232] Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims. Allcited references referred to in the application are incorporated byreference.

What is claimed:
 1. A method for blocking the interaction of an integrinwith a cytoplasmic signaling partner comprising the step of contactingan integrin having a phosphorylated tyrosine in the cytoplasmic domainof the β subunit, or contacting a fragment thereof comprising thephosphorylated cytoplasmic domain, with an agent which blocks thebinding of said signaling partner to said integrin.
 2. The method ofclaim 1 wherein said agent blocks the binding of said integrin to saidsignaling partner by selectively binding to the phosphorylatedcytoplasmic domain of the β subunit of said integrin.
 3. The method ofclaim 1 wherein said agent blocks the binding of said integrin to saidsignaling partner by selectively binding to said signaling partner. 4.The method of claim 3 wherein said agent is a phosphorylated peptide. 5.The method of claim 4 wherein said phosphorylated peptide has an aminoacid sequence selected from the group consisting of the sequencespresented in Examples 2 and 4-8 of this specification, together withfragments and variants.
 6. The method of claim 1 wherein said integrincomprises a β subunit selected from the group consisting of the β-1,β-2, β-3, β-5, β-6 and β-7 subunit.
 7. The method of claim 1 whereinsaid signaling partner is selected from the group consisting of thesrc-family of tyrosine kinases and the non-src family of tyrosinekinases.
 8. The method of claim 7 wherein said tyrosine kinase isselected from the group consisting of the p60c-src, p56lyn, p59fyntyrosine kinase, Gnb2 and Shc.
 9. The method of claim 1 wherein saidblocking reduces cellular aggregation of an integrin expressing cell.10. The method of claim 1 wherein said blocking reduces cellularattachment of an integrin expressing cell.
 11. The method of claim 1wherein said blocking reduces cellular migration of an integrinexpressing cell.
 12. A method for reducing the severity of pathologicalstate mediated by integrin signaling comprising the method of claim 1.13. The method of claim 12 wherein said pathological state is selectedfrom the group consisting of thrombosis, inflammation, and tumormetastasis.
 14. A method for identifying agents which block theinteraction of an integrin with a cytoplasmic signaling partnercomprising the steps of: a) incubating a peptide comprising thephosphorylated cytoplasmic domain of the β subunit of said integrin withsaid signaling partner and with an agent, and b) determining whethersaid agent blocks the binding of said signaling partner to said peptide.15. The method of claim 14 wherein said peptide comprising thephosphorylated cytoplasmic domain of the β subunit of said integrin isselected from the group consisting of the β-1, β-2, β-3, β-5, β-6, andβ-7 subunit.
 16. The method of claim 14 wherein said peptide comprisingthe phosphorylated cytoplasmic domain of the β subunit of said integrincomprises an amino acid sequence selected from the group consisting ofthe sequences presented in Examples 2 and 4-8 of this specification,together with fragments and variants.
 17. The method of claim 14 whereinsaid signaling partner is contained in an extract of a cell whichexpresses an integrin having a phosphorylated tyrosine in thecytoplasmic domain of the subunit.
 18. The method of claim 17 whereinsaid extract of a cell is prepared from a cell selected from the groupconsisting of platelets and leukocytes.
 19. The method of claim 18wherein said cell is activated prior to the preparation of said cellextract.
 20. The method of claim 19, wherein said platelets areactivated with thrombin.
 21. A method to identify integrin mediatedsignaling comprising the step of determining whether the cytoplasmicdomain of said integrin is phosphorylated.
 22. The method of claim 21comprising the steps of; a) preparing an extract of a cell expressing anintegrin, b) electrophoresing said extract using SDS electrophoresis,and c) analyzing said electrophoresed sample to determine whether the βsubunit of said integrin is phosphorylated.
 23. The method of claim 22wherein an anti-phosphotyrosine antibody is used in the analysis stepc).
 24. A method to identify an integrin signaling partner comprisingthe steps a) preparing an extract from a cell which expresses anintegrin, b) incubating said extract with a peptide comprising thephosphorylated cytoplasmic domain of the β subunit of an integrin, andc) separating phosphorylated cytoplasmic domain of the β subunit whichbound said signaling partner from the mixture of step (b).
 25. Themethod of claim 22, wherein said phosphorylated cytoplasmic domain hasan amino acid sequence selected from the group consisting of thesequences presented in Examples 2 and 4-8 of this specification,together with fragments and variants.
 26. The method of claim 23 whereinsaid phosphorylated cytoplasmic domain is immobilized on a solidsupport.
 27. An isolated peptide consisting essentially of an amino acidsequence selected from the group consisting of the sequences presentedin Examples 2 and 4-8 of this specification, together with fragments andvariants.
 28. The peptide of claim 27 wherein one or more of thetyrosine residues in said amino acid sequence is irreversiblyphosphorylated.
 29. A method for treating pathological conditions,comprising: the administration of the peptide of claim 27, wherein thecondition is selected from the group consisting of acute coronarysyndrome, myocardial infarction, unstable angina, refractory angina,occlusive coronary thrombus occurring post-thrombolytic therapy orpost-coronary angioplasty, a thrombotically mediated cerebrovascularsyndrome, embolic stroke, thrombotic stroke, transient ischemic attacks,venous thrombosis, deep venous thrombosis, pulmonary embolus,coagulopathy, disseminated intravascular coagulation, thromboticthrombocytopenic purpura, thromboangiitis obliterans, thrombotic diseaseassociated with heparin-induced thrombocytopenia, thromboticcomplications associated with extracorporeal circulation, thromboticcomplications associated with instrumentation such as cardiac or otherintravascular catheterization, intra-aortic balloon pump, coronary stentor cardiac valve, and conditions requiring the fitting of prostheticdevices.